CN101620173A - Homocysteine diagnosis kit and method for determining homocysteine concentration - Google Patents
Homocysteine diagnosis kit and method for determining homocysteine concentration Download PDFInfo
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- CN101620173A CN101620173A CN200810124243A CN200810124243A CN101620173A CN 101620173 A CN101620173 A CN 101620173A CN 200810124243 A CN200810124243 A CN 200810124243A CN 200810124243 A CN200810124243 A CN 200810124243A CN 101620173 A CN101620173 A CN 101620173A
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Abstract
The invention relates to a homocysteine diagnosis kit using the technology of an enzyme colorimetric method and an ELISA method, a method for determining homocysteine concentration, and composition and components of a reagent, and belongs to the technical field of medical test and determination. The kit comprises the following main components: buffer solution, reduced coenzyme, 2-ketogulataric acid, homocysteine-alpha, gamma-lyase, glutamic dehydrogenase and a stabilizer. The method for determining homocysteine concentration comprises the following steps: mixing a sample and the reagent according to a certain volume ratio to perform a series of enzymic reaction, and then placing reactants under a UV/ visible light analyzer to test the descending speed of absorbance at the position where the dominant wave length is 340nm to determine the concentration of homocysteine.
Description
Technical field
The present invention relates to a kind of homotype semicystionl diagnostic kit, 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 concentration 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 etc.
High performance liquid chromatography (HPLC): be classical reference method, but its operation more complicated, test consuming time longly that the test figure variance ratio is bigger, is replaced by enzyme-linked immunosorbent assay, fluorescence polarization assay method and enzyme process gradually aspect clinical practice.High performance liquid chromatography: 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.
Enzyme-linked immunosorbent assay (EIA): be the common method of measuring homocysteine level clinically, operate more convenient, quick, good reproducibility, method is reliable and stable.Its shortcoming is most of operation or manual operations, and is more consuming time.Enzyme-linked immunosorbent assay fundamental analysis principle: 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.
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.
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, its operation more complicated, the test long and bigger shortcoming of test figure variance ratio consuming time.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.
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.
Enzyme process: the method for testing of coming out newly developed in response to the market demand, at present domestic have a multinomial patented claim, and CN98807531.8 utilizes 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 measurable fluorescent chemicals 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 (Enzymatic ColorimetricMethod) 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 realize the homotype semicystionl diagnostic kit of this method, 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 principle of the present invention is as follows:
Homocysteine+water
Homocysteine-alpha, gamma-lyasesSulfuretted hydrogen+ammonia+
The alpha-oxo-butyric acid
Ammonia+2-oxoglutaric acid+reduced coenzyme
Glutamte dehydrogenaseGlutamic acid+coenzyme+
Water
This method is used homocysteine-alpha, gamma-lyases (homocysteine desulfhydrase; EC4.4.1.2) enzyme (idol) connection glutamte dehydrogenase (Glutamate Dehydrogenase; EC 1.4.1.2; EC 1.4.1.3; EC 1.4.1.4) enzymatic reaction colourimetry.Homocysteine-α, γ-lyases enzymolysis homocysteine reaction produces ammonia, the effect of uniting glutamte 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 the homotype semicystionl diagnostic kit of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Homocysteine-alpha, gamma-lyases 9000U/L
Glutamte dehydrogenase 8000U/L
2-oxoglutaric acid 15mmol/L
Homotype semicystionl diagnostic kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, homocysteine-alpha, gamma-lyases, glutamte dehydrogenase, 2-oxoglutaric 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.
Also above-mentioned single agent reagent can be made into following pair of agent reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme, 2-oxoglutaric acid.
Reagent 2
Damping fluid, stabilizing agent, homocysteine-alpha, gamma-lyases, glutamte dehydrogenase.
Reduced coenzyme, homocysteine-alpha, gamma-lyases, glutamte dehydrogenase, the position of 2-oxoglutaric acid 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.
Above-mentioned single agent reagent can also be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme, 2-oxoglutaric acid.
Reagent 2
Damping fluid, stabilizing agent, glutamte dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, homocysteine-alpha, gamma-lyases.
Reduced coenzyme, homocysteine-alpha, gamma-lyases, glutamte dehydrogenase, the position of 2-oxoglutaric acid 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 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 reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Homocysteine-alpha, gamma-lyases 9000U/L
Glutamte dehydrogenase 8000U/L
2-oxoglutaric acid 15mmol/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 reagent of present embodiment is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
2-oxoglutaric acid 15mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Homocysteine-alpha, gamma-lyases 9000U/L
Glutamte dehydrogenase 8000U/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 reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
2-oxoglutaric acid 15mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glutamte dehydrogenase 8000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Homocysteine-alpha, gamma-lyases 9000U/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)≤0.0007; Absorbance time response curve should be decline curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 1mmol/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 0.3 ± 0.15 Δ A/mmol/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 principle is as follows:
Homocysteine+water
Homocysteine-alpha, gamma-lyasesSulfuretted hydrogen+ammonia+
The alpha-oxo-butyric acid
Ammonia+2-oxoglutaric acid+reduced coenzyme
Glutamte dehydrogenaseGlutamic acid+coenzyme+
Water
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. homotype semicystionl diagnostic kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
Reduced coenzyme 0.1---0.35mmol/L
Homocysteine-alpha, gamma-lyases 1000---80000U/L
Glutamte dehydrogenase 1000---80000U/L
2-oxoglutaric acid 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: 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:
Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, homocysteine-alpha, gamma-lyases, glutamte dehydrogenase, 2-oxoglutaric acid.
4. according to the described homotype semicystionl diagnostic kit of claim 2, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, homocysteine-alpha, gamma-lyases, glutamte dehydrogenase, 2-oxoglutaric acid; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, 2-oxoglutaric acid; Reagent 2 is made up of damping fluid, stabilizing agent, homocysteine-alpha, gamma-lyases, glutamte dehydrogenase.Reduced coenzyme, homocysteine-alpha, gamma-lyases, glutamte dehydrogenase, the position of 2-oxoglutaric acid in reagent 1 or reagent 2 can not limit.
5. according to the described homotype semicystionl diagnostic kit of claim 2, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, homocysteine-alpha, gamma-lyases, glutamte dehydrogenase, 2-oxoglutaric acid; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, 2-oxoglutaric acid; Reagent 2 is made up of damping fluid, stabilizing agent, glutamte dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, homocysteine-alpha, gamma-lyases.Reduced coenzyme, homocysteine-alpha, gamma-lyases, glutamte dehydrogenase, the position of 2-oxoglutaric acid in reagent 1, reagent 2 or reagent 3 can not limit.
6. 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 (AmmoniaSulfate), 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 |
|---|---|---|---|---|
| CN106198995A (en) * | 2016-06-24 | 2016-12-07 | 常州英赞美科生物科技有限公司 | A kind of homotype semicystionl diagnostic kit and the assay method of homotype semicystinol concentration investigating |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106198995A (en) * | 2016-06-24 | 2016-12-07 | 常州英赞美科生物科技有限公司 | A kind of homotype semicystionl diagnostic kit and the assay method of homotype semicystinol concentration investigating |
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Open date: 20100106 |