CN101750396A - Homocysteine diagnosing/measuring reagent (kit) and homocysteine concentration measuring method - Google Patents
Homocysteine diagnosing/measuring reagent (kit) and homocysteine concentration measuring method Download PDFInfo
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- CN101750396A CN101750396A CN200810244299A CN200810244299A CN101750396A CN 101750396 A CN101750396 A CN 101750396A CN 200810244299 A CN200810244299 A CN 200810244299A CN 200810244299 A CN200810244299 A CN 200810244299A CN 101750396 A CN101750396 A CN 101750396A
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- methionine
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Abstract
The invention relates to a homocysteine diagnosing/measuring reagent (kit) using the technologies of an enzyme colorimetric method and an enzyme linkage method. At the same time, the invention also relates to a homocysteine concentration measuring method, the reagent composition and reagent ingredients, which belong to the technical field of medical detection and measurement. The reagent (kit) mainly comprises the following ingredients: a buffer solution, reduced form cozymase, adenosylmethionine, oxyacid, homocysteine methyltransferase, methionine gama-lyase, amino acid dehydrogenase and stabilizing agents. The method has the following steps: mixing samples and the reagent by the certain volume percent for making the samples and the reagent take a series of enzymatic reaction, and then, placing reactants under an ultraviolet/visible light analyzer to detect the dropping degree of the light absorbancy in the position with the main wave length of 340 nm, so the concentration of the homocysteine can be measured and calculated.
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, amino acid 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 enzymic colorimetric (EnzymaticColorimetric Method) and enzyme (even) united method (Couple Reaction) technology utilized, 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+adenyl residue methionine homocysteine methyltransgerase
Adenyl residue homocysteine+methionine
Methionine+water methionine Gamma-catenase methyl mercaptan+ammonia+2-ketobutyric acid
Ammonia+oxyacid+reduced coenzyme amino acid deoxygenase amino acid+water+coenzyme
This method is used homocysteine methyltransgerase (homocysteine S-methyltransferase; EC 2.1.1.10) enzyme (idol) connection methionine Gamma-catenase (methionine γ-lyase; EC 4.4.1.11), amino acid dehydrogenase (amino-acid dehydrogenase; EC 1.4.1.5; EC 1.4.99.1) enzymatic reaction colorimetric end-point method.The reaction of homocysteine methyltransgerase enzymolysis homocysteine produces methionine, the effect of uniting methionine Gamma-catenase, amino acid 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 degree that reduced coenzyme descends in 340nm place absorbance, by measuring the degree 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 methyltransgerase 12000U/L
Methionine Gamma-catenase 9000U/L
Amino acid dehydrogenase 10000U/L
Adenyl residue methionine 9mmol/L
Oxyacid 12mmol/L
Homocysteine diagnosis of the present invention/mensuration reagent (box) can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, homocysteine methyltransgerase, methionine Gamma-catenase, amino acid dehydrogenase, adenyl residue methionine, oxyacid.
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, adenyl residue methionine, oxyacid.
Reagent 2
Damping fluid, stabilizing agent, homocysteine methyltransgerase, methionine Gamma-catenase, amino
Acidohydrogenase.
Reduced coenzyme, homocysteine methyltransgerase, methionine Gamma-catenase, amino acid dehydrogenase, adenyl residue methionine, the position of oxyacid 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, adenyl residue methionine, oxyacid.
Reagent 2
Damping fluid, stabilizing agent, methionine Gamma-catenase, amino acid dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, homocysteine methyltransgerase.
Reduced coenzyme, homocysteine methyltransgerase, methionine Gamma-catenase, amino acid dehydrogenase, adenyl residue methionine, the position of oxyacid 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 methyltransgerase 12000U/L
Methionine Gamma-catenase 9000U/L
Amino acid dehydrogenase 10000U/L
Adenyl residue methionine 9mmol/L
Oxyacid 12mmol/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 degree 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
Adenyl residue methionine 9mmol/L
Oxyacid 12mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Homocysteine methyltransgerase 12000U/L
Methionine Gamma-catenase 9000U/L
Amino acid 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 degree 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
Adenyl residue methionine 9mmol/L
Oxyacid 12mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Methionine Gamma-catenase 9000U/L
Amino acid dehydrogenase 10000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Homocysteine methyltransgerase 12000U/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 degree 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 600l 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 0.0004 ± 0.0002 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 enzymic colorimetric and enzyme-linked method technology, its method is as follows:
Homocysteine+adenyl residue methionine
Homocysteine methyltransgerase
Adenyl residue homocysteine+methionine
Methionine+water
Methionineγ
-lyasesMethyl mercaptan+ammonia+2-ketobutyric acid
Ammonia+oxyacid+reduced coenzyme
Amino acid dehydrogenaseAmino acid+water+coenzyme
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the degree 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 methyltransgerase 1000---80000U/L
Methionine Gamma-catenase 1000---80000U/L
Amino acid dehydrogenase 1000---80000U/L
Adenyl residue methionine 1---50mmol/L
Oxyacid 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 methyltransgerase, methionine Gamma-catenase, amino acid dehydrogenase, adenyl residue methionine, oxyacid.
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 methyltransgerase, methionine Gamma-catenase, amino acid dehydrogenase, adenyl residue methionine, oxyacid; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, adenyl residue methionine, oxyacid; Reagent 2 is made up of damping fluid, stabilizing agent, homocysteine methyltransgerase, methionine Gamma-catenase, amino acid dehydrogenase.Reduced coenzyme, homocysteine methyltransgerase, methionine Gamma-catenase, amino acid dehydrogenase, adenyl residue methionine, the position of oxyacid 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 methyltransgerase, methionine Gamma-catenase, amino acid dehydrogenase, adenyl residue methionine, oxyacid; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, adenyl residue methionine, oxyacid; Reagent 2 is made up of damping fluid, stabilizing agent, methionine Gamma-catenase, amino acid dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, homocysteine methyltransgerase.Reduced coenzyme, homocysteine methyltransgerase, methionine Gamma-catenase, amino acid dehydrogenase, adenyl residue methionine, the position of oxyacid 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 |
|---|---|---|---|---|
| CN102298025A (en) * | 2010-06-25 | 2011-12-28 | 苏州艾杰生物科技有限公司 | Method for determining glycine and glycine determination kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102298025A (en) * | 2010-06-25 | 2011-12-28 | 苏州艾杰生物科技有限公司 | Method for determining glycine and glycine determination kit |
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Application publication date: 20100623 |