CN1757755B - Determination method of creatnine content and reagent box for diagnosing creatnine - Google Patents
Determination method of creatnine content and reagent box for diagnosing creatnine Download PDFInfo
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- CN1757755B CN1757755B CN 200410064891 CN200410064891A CN1757755B CN 1757755 B CN1757755 B CN 1757755B CN 200410064891 CN200410064891 CN 200410064891 CN 200410064891 A CN200410064891 A CN 200410064891A CN 1757755 B CN1757755 B CN 1757755B
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 29
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 claims abstract description 108
- 229940109239 creatinine Drugs 0.000 claims abstract description 55
- 239000005515 coenzyme Substances 0.000 claims abstract description 46
- 230000002829 reductive effect Effects 0.000 claims abstract description 24
- 239000000872 buffer Substances 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims description 28
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 claims description 28
- 238000013016 damping Methods 0.000 claims description 26
- 239000012530 fluid Substances 0.000 claims description 26
- -1 2-oxoglutaric acid ester Chemical class 0.000 claims description 20
- 108010029444 creatinine deiminase Proteins 0.000 claims description 18
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 claims description 17
- 102000016901 Glutamate dehydrogenase Human genes 0.000 claims description 17
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 17
- 229960003624 creatine Drugs 0.000 claims description 14
- 239000006046 creatine Substances 0.000 claims description 14
- 238000003745 diagnosis Methods 0.000 claims description 14
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 12
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 11
- 230000035484 reaction time Effects 0.000 claims description 9
- 229960003966 nicotinamide Drugs 0.000 claims description 8
- 235000005152 nicotinamide Nutrition 0.000 claims description 8
- 239000011570 nicotinamide Substances 0.000 claims description 8
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 6
- 230000001186 cumulative effect Effects 0.000 claims description 6
- 229930195712 glutamate Natural products 0.000 claims description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 6
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 claims description 4
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 claims description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 4
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 claims description 4
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 3
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 3
- 229960004418 trolamine Drugs 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 150000005846 sugar alcohols Polymers 0.000 claims description 2
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 claims 2
- 229940058020 2-amino-2-methyl-1-propanol Drugs 0.000 claims 1
- 108010008488 Glycylglycine Proteins 0.000 claims 1
- CBTVGIZVANVGBH-UHFFFAOYSA-N aminomethyl propanol Chemical compound CC(C)(N)CO CBTVGIZVANVGBH-UHFFFAOYSA-N 0.000 claims 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims 1
- 229940043257 glycylglycine Drugs 0.000 claims 1
- WVKIFIROCHIWAY-UHFFFAOYSA-N hydron;2-(methylamino)acetic acid;chloride Chemical compound Cl.CNCC(O)=O WVKIFIROCHIWAY-UHFFFAOYSA-N 0.000 claims 1
- 239000002953 phosphate buffered saline Substances 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 19
- 102000004190 Enzymes Human genes 0.000 abstract description 6
- 108090000790 Enzymes Proteins 0.000 abstract description 6
- 238000005859 coupling reaction Methods 0.000 abstract description 5
- 238000002156 mixing Methods 0.000 abstract description 2
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 abstract 1
- 239000007788 liquid Substances 0.000 abstract 1
- 230000003287 optical effect Effects 0.000 abstract 1
- 239000003381 stabilizer Substances 0.000 abstract 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 28
- 229910021529 ammonia Inorganic materials 0.000 description 14
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 5
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 4
- 108010082126 Alanine transaminase Proteins 0.000 description 4
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 4
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 235000011089 carbon dioxide Nutrition 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 108010066906 Creatininase Proteins 0.000 description 2
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- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 2
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- VXEQGQXRKQSAMW-UHFFFAOYSA-N 2-amino-2-methylpropan-1-ol Chemical compound CC(C)(N)CO.CC(C)(N)CO VXEQGQXRKQSAMW-UHFFFAOYSA-N 0.000 description 1
- GNWUOVJNSFPWDD-XMZRARIVSA-M Cefoxitin sodium Chemical compound [Na+].N([C@]1(OC)C(N2C(=C(COC(N)=O)CS[C@@H]21)C([O-])=O)=O)C(=O)CC1=CC=CS1 GNWUOVJNSFPWDD-XMZRARIVSA-M 0.000 description 1
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- PGXABRVTTLRYCN-UHFFFAOYSA-N Cl.Cl.Cl.Cl.C(=O)(O)CCN Chemical compound Cl.Cl.Cl.Cl.C(=O)(O)CCN PGXABRVTTLRYCN-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A method for measuring the content of creatinine by use of the creatinine testing reagent kit includes such steps as proportionally mixing said reagents with specimen, enzyme coupling reaction, taking the resultant, detecting the variation of primary wave in optical absorbancy by biochemical analyzer and calculating the content of creatinine. Said reagent kit is composed of 6 reagents including buffer liquid, 2-ketoglutarate, reductive coenzyme, stabilizer, etc.
Description
Technical field
The present invention relates to a kind of method of measuring creatinine content, the invention still further relates to the creatine diagnosis reagent kit that is used to realize this method simultaneously, belong to medical test determination techniques field.
Background technology
Measure creatinine and mainly contain chemical assay (Jaffe method), enzyme process, high performance liquid chromatography (HPLC) and capillary electrophoresis etc.
Chemical assay---with low cost, easy and simple to handle, be one of the most frequently used method of present domestic mensuration creatinine.Creatinine in the sample and picrate effect generate the picric acid creatinine mixture of yellowish red color.The shortcoming of this method is that specificity is not high, because vitamins C, acetone, etheric acid, methyldopa and high concentration glucose, protein and some microbiotic such as penicillin G, cefoxitin, Kefzol etc. also can generate red with the alkaline picric acid reaction.
High performance liquid chromatography (HPLC) (HPLC)---creatinine is positively charged in weak acid environment, can separate with other compositions are fine by the cation-exchange chromatography post, measures its photoabsorption at 234nm.Precision height, specificity that this method is analyzed are good, but this law is unsuitable for clinical samples analysis in enormous quantities, usually only as the reference method of creatinine assay, are used to estimate test kit and some scientific research purpose of commercially available creatinine assay.
Capillary electrophoresis---serum specimen is done pre-treatment with high speed centrifugation, and urine specimen can be used low-speed centrifugal, removes formed elements, and supernatant liquor is measured 235nm place absorbancy after moving the electrocapillary electrophoretic separation with micella.It is wide that this law is measured linearity range, operate comparatively easy, but need with specific installation with carry out the pre-treatment of serum specimen, routine clinical use difficulty.
The enzymatic determination method---mainly contain Creatinine deiminase (Creatinine deiminase) and Creatininase (Creatininase) two big classes.Retrieval finds, the patent application that application number is 02139298.6, the applying date is 2002.11.15 discloses the mensuration reagent of using the preparation of enzyme reaction product oxidized form nicotinamide coenzyme.The enzymic measuring reagent of this invention indication does not add the required reduced form nicotinamide coenzyme of assaying reaction or its analogue, and add its reaction product oxidized form nicotinamide coenzyme or its analogue and generate required enzyme of reduced coenzyme and substrate system accordingly, by in reagent, forming long response time systems such as enzyme-substrate-NAD or enzyme-substrate-NADP, this class oxidized coenzyme or the slow circulation of its analogue are reduced to reduced form nicotinamide coenzyme or its analogue.When the reduced form nicotinamide coenzyme that is reduced or its analogue reached certain concentration, the enzyme process reagent of this invention indication just can reach the purpose of measuring alanine aminotransferase, aspartate amino transferase, urea, ammonia, creatinine and carbonic acid gas etc. in the sample.This characteristic feature of an invention is that the test of supporting reagent, ammonia reagent, creatinine reagent and carbonic acid gas reagent etc. for alanine aminotransferase reagent, aspartate amino transferase reagent, urea provides an endogenous synthetic alanine aminotransferase reagent, aspartate amino transferase reagent, urea to support reaction needed substrate-reduced form nicotinamide coenzymes such as reagent, ammonia reagent, creatinine reagent and carbonic acid gas reagent.One of its shortcoming is, this system is in the needed reduced form nicotinoyl of formation reaction ammonia coenzyme, also offset the activity of part alanine aminotransferase, aspartate amino transferase, urea, ammonia, creatinine and carbonic acid gas etc., caused the accuracy of reagent test to reduce greatly.Two of its shortcoming is that this reagent must react for some time before official testing in advance, so that can produce enough reduced form nicotinoyl ammonia coenzyme, has so just caused the result that slow action cannot save a critical situation, brings very big inconvenience to test.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of measuring method that can overcome the creatinine content of above prior art shortcoming, provide the creatine diagnosis reagent kit of this method of realization simultaneously, adopt the reagent in this test kit not only can on ultraviolet analyser or half, automatic clinical chemistry analyzer, carry out creatinine content determination, and finding speed is fast, accuracy is high, thereby can obtain practical applying.
It is as follows that the present invention measures the method steps of creatinine content:
1), with sample and the reagent mix of mainly forming by 2-oxoglutaric acid ester, reducibility coenzyme, Creatinine deiminase, glutamate dehydrogenase, make it to take place the reaction of following principle:
Creatinine+water+H
+ Creatinine deiminaseN-methyl hydantoin+ammonium ion
Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme
Glutamate dehydrogenaseL-glutamic acid+coenzyme
2), detect the end reaction thing in the speed that predominant wavelength 340nm absorbancy descends, calculate the size of creatinine content.
Common step 2) the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer device, detect the speed that predominant wavelength 340nm absorbancy descends, calculate the size of creatinine content.
The blending ratio of above sample and reagent by volume is 1/10 to 1/250, and the mensuration temperature of above process is controlled at 20 ℃ to 50 ℃ conventional scopes, and the reaction times was controlled at conventional 2-30 minute.
The present invention uses Creatinine deiminase coupling glutamate dehydrogenase reaction continuous monitoring method or colorimetry.Creatinine deiminase produces ammonia with the creatinine deamination, and the effect by the coupling glutamate dehydrogenase again is oxidized into coenzyme (NAD with reduced coenzyme (NADH, NADPH or other analogue---absorption peak is arranged at the 340nm place)
+, NADP
+Or other analogue---do not have absorption peak at the 340nm place), thereby measured speed (journey) degree that reduced coenzyme descends in 340nm place absorbancy, by measuring speed (journey) degree that 340nm place absorbancy descends, can calculate the content of creatinine.
Be used to realize that the creatine diagnosis reagent kit of the inventive method can be single agent, comprise:
Damping fluid 40---200mmol/l
2-oxoglutaric acid ester 0.5---50mmol/l
Reducibility coenzyme 0.15---0.5mmo/l
Creatinine deiminase 1000---50000U/l
Glutamate dehydrogenase 5000---90000U/l
Stablizer 10---80% (cumulative volume)
Also above single agent can be made into following pair of agent, more help eliminating inside and outside source ammonia pollution:
Reagent I
Damping fluid 40---200mmol/l
2-oxoglutaric acid ester 0.5---50mmol/l
Reducibility coenzyme 0.15---0.5mmo/l
Glutamate dehydrogenase 5000---90000U/l
Stablizer 10---80% (cumulative volume)
Reagent II
Damping fluid 40---200mmol/l
Creatinine deiminase 1000---50000U/l
Stablizer 10---80% (cumulative volume)
If do not consider inside and outside source ammonia pollution, the prescription of two agent is not limited only to above-mentioned prescription, the composition of reagent I wherein, glutamate dehydrogenase, 2-oxoglutaric acid ester or reducibility coenzyme etc. can be placed on reagent II, reagent II composition wherein, Creatinine deiminases etc. also can be placed on reagent I, so can form multiple formulations, do not describe in detail one by one.
Reagent can also be made into following three reagent, not only more help eliminating inside and outside source ammonia pollution, also more help the stable of reagent:
Reagent I
Damping fluid 40---200mmol/l
2-oxoglutaric acid ester 0.5---50mmol/l
Reducibility coenzyme 0.15---0.5mmo/l
Stablizer 10---50mmo/l
Reagent II
Damping fluid 40---200mmol/l
Glutamate dehydrogenase 5000---90000U/l
Stablizer 10---80% (cumulative volume)
Reagent III
Damping fluid 40---200mmol/l
Creatinine deiminase 1000---50000U/l
Stablizer 10---80% (cumulative volume)
Three doses prescription is not limited only to above-mentioned prescription, the composition of reagent I wherein, and 2-oxoglutaric acid ester, reducibility coenzyme etc. can be placed among the reagent II, reagent II composition wherein, glutamate dehydrogenase also can be placed among the reagent I, so can form multiple formulations, does not describe in detail one by one at this.
The pH scope of buffer reagent can be 6.0-11.0.Buffer reagent can be three (carboxymethyl) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, phosphoric acid salt (Phosphate) damping fluid, trolamine (Triethanolamine) damping fluid, 2-amino-2-methyl-1-propanol (2-Amino-2-methyl-1-propanol) damping fluid, imidazoles (Imidazole) damping fluid or glycylglycine (Glycylglycine) damping fluid etc., but is not limited only to these damping fluids.
Above reduced coenzyme can be reduced form nicotinamide coenzyme or derivatives thereofs such as NADPH, NADH or thio-NADH.
In addition, in order to reduce the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stablizer 10-80% or 10-50mmol/l among reagent I, the reagent II of the reagent I of above single agent, two agent, reagent II or three doses, the reagent III, stablizer can be one or more in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, salt or the adenosine diphosphate (ADP) etc.
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 creatine diagnosis reagent kit of the present invention of following system component relation is comparatively desirable:
Damping fluid 80---120mmol/l
2-oxoglutaric acid ester 5---20mmol/l
Reducibility coenzyme 0.15---0.3mmo/l
Creatinine deiminase 2000---20000U/l
Glutamate dehydrogenase 10000---50000U/l
Stablizer 20---50%
Because the present invention utilizes Enzymology method, enzyme digestion reaction to have the high characteristics of specificity fully, is not vulnerable to the interference of other materials of inside and outside source.Easy, the easy handling of enzyme process method.The specificity of enzyme digestion reaction impels test result accurate.Enzyme digestion reaction all is to carry out under buffer conditions, does not have environmental pollution problem.The enzyme process method does not need special, extra instrument, and testing cost is cheap.Therefore can guarantee to have higher test accuracy, be more convenient for applying.
In addition, one of striking features of the present invention is the pollution that can eliminate inside and outside source ammonia, the effect of eliminating inside and outside source ammonia occurs in the first half of entire reaction time period, be consumed totally at time second half section contaminated inside and outside source ammonia, and all be the content that results from creatinine at the needed ammonia of second half section time test creatinine content.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one (single agent)
The creatine diagnosis reagent kit of present embodiment comprises:
Damping fluid 80mmol/l
2-oxoglutaric acid ester 5mmol/l
Reducibility coenzyme 0.15mmo/l
Creatinine deiminase 2000U/l
Glutamate dehydrogenase 10000U/l
Stablizer 50%
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested creatinine sample and reagent is 1/25, the Direction of Reaction is negative reaction.
After adding sample and reagent, make it to mix, following reaction take place:
Creatinine+water+H
+ Creatinine deiminaseN-methyl hydantoin+ammonium ion
Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme
Glutamate dehydrogenaseL-glutamic acid+coenzyme
The end reaction thing is placed under the biochemical analyzer, detect speed (journey) degree that predominant wavelength 340nm absorbancy descends, thereby calculate the content of creatinine.
Present embodiment is used Creatinine deiminase coupling glutamate dehydrogenase reaction continuous monitoring method or colorimetry.Creatinine deiminase produces ammonia with the creatinine deamination, effect by the coupling glutamate dehydrogenase again, reduced coenzyme is oxidized into coenzyme, thereby measured speed (journey) degree that reduced coenzyme descends in 340nm place absorbancy, by measuring speed (journey) degree that 340nm place absorbancy descends, can calculate the content of creatinine.
Embodiment two (two agent)
The creatine diagnosis reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
2-oxoglutaric acid ester 12mmol/l
Reducibility coenzyme 0.25mmo/l
Glutamate dehydrogenase 30000U/l
Stablizer 50%
Reagent II
Damping fluid 100mmol/l
Creatinine deiminase 11000U/l
Stablizer 50%
When measuring creatinine content, temperature is controlled at 30 ℃, 15 minutes reaction times, and test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested creatinine sample and reagent is 1/25, the Direction of Reaction is negative reaction.
Concrete determination step is:
Creatinine+water+H
+ Creatinine deiminaseN-methyl hydantoin+ammonium ion
Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme
Glutamate dehydrogenaseL-glutamic acid+coenzyme
The end reaction thing is placed under the biochemical analyzer, detect speed (journey) degree that predominant wavelength 340nm absorbancy descends, thereby calculate the content of creatinine.
The reaction times of each reactions steps was controlled at 15 minutes.
Embodiment three (three doses)
The creatine diagnosis reagent of present embodiment is three doses, has:
Reagent I
Damping fluid 120mmol/l
2-oxoglutaric acid ester 20mmol/l
Reducibility coenzyme 0.3mmo/l
Stablizer 20mmo/l
Reagent II
Damping fluid 120mmol/l
Glutamate dehydrogenase 50000U/l
Stablizer 50%
Reagent III
Damping fluid 120mmol/l
Creatinine deiminase 20000U/l
Stablizer 50%
On automatic clinical chemistry analyzer, set: 25 ℃ of temperature, 20 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested creatinine sample and reagent is 1/25, the Direction of Reaction is negative reaction.
Concrete determination step is:
Creatinine+water+H
+ Creatinine deiminaseN-methyl hydantoin+ammonium ion
Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme
Glutamate dehydrogenaseL-glutamic acid+coenzyme
The end reaction thing is placed under the biochemical analyzer, detect speed (journey) degree that predominant wavelength 340nm absorbancy descends, thereby calculate the content of creatinine.
The reaction times of each reactions steps was controlled at 20 minutes.
Embodiment four
The creatine diagnosis reagent of present embodiment has:
Reagent I
Damping fluid 100mmol/l
2-oxoglutaric acid ester 10mmol/l
Reducibility coenzyme 0.25mmo/l
Glutamate dehydrogenase 50000U/l
Stablizer 50%
Reagent II
Damping fluid 100mmol/l
Creatinine deiminase 8000U/l
Stablizer 50%
On Biochemical Analyzer, set: temperature 37,10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of tested creatinine sample and reagent is 1/25, the Direction of Reaction is negative reaction.
After adding sample and reagent, make it to mix, following reaction take place:
Creatinine+water+H
+ Creatinine deiminaseN-methyl hydantoin+ammonium ion
Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme
Glutamate dehydrogenaseL-glutamic acid+coenzyme
The end reaction thing is placed under the biochemical analyzer, detect speed (journey) degree that predominant wavelength 340nm absorbancy descends, thereby calculate the content of creatinine.
In a word, experiment showed, and adopt measuring method of the present invention can draw required measurement result by ultraviolet analyser or half, automatic clinical chemistry analyzer device fully, and highly sensitive, tolerance range good, is not subjected to the pollution of inside and outside source material.
Claims (9)
1. creatinine content determination method, step is as follows:
1), with sample and the reagent mix of mainly forming by 2-oxoglutaric acid ester, reducibility coenzyme, Creatinine deiminase, glutamate dehydrogenase, make it to take place the reaction of following principle:
Creatinine+water+H
+ Creatinine deiminaseN-methyl hydantoin+ammonium ion
Ammonium ion+2-oxoglutaric acid ester+reduced coenzyme
Glutamate dehydrogenaseL-glutamic acid+coenzyme
2), detect the end reaction thing in the speed that predominant wavelength 340nm absorbancy descends, calculate the size of creatinine content.
2. according to the described creatinine content determination method of claim 1, it is characterized in that: described step 2) be: the end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the degree that predominant wavelength 340nm absorbancy descends, calculate the content of creatinine.
3. according to claim 1 or 2 described creatinine content determination methods, it is characterized in that: temperature is controlled at 20 ℃ to 50 ℃ scopes, and the reaction times was controlled at 2-30 minute.
4. according to claim 1 or 2 described creatinine content determination methods, it is characterized in that: the ratio control of tested creatinine sample and reagent is 1/10 to 1/500.
5. creatine diagnosis reagent kit is grouped into by following one-tenth:
Damping fluid 40---200mmol/l
2-oxoglutaric acid ester 0.5---50mmol/l
Reducibility coenzyme 0.15---0.5mmo/l
Creatinine deiminase 1000---50000U/l
Glutamate dehydrogenase 5000---90000U/l
Stablizer 10---80% cumulative volume ratio
6. according to the described creatine diagnosis reagent kit of claim 5, it is characterized in that: described reagent is made into single agent, two agent or three doses.
7. according to claim 5 or 6 described creatine diagnosis reagent kits, it is characterized in that: described stablizer is at least a in ethylene glycol, propylene glycol, glycerine, glycan, polyalcohols, salt or the adenosine diphosphate (ADP).
8. according to claim 5 or 6 described creatine diagnosis reagent kits, it is characterized in that: the pH scope of described buffer reagent is 6.0-11.0, and described buffer reagent is a kind of in three carboxymethylamino methane-hydrochloride buffers, phosphate buffered saline buffer, trolamine damping fluid, 2-amino-2-methyl-1-propanol damping fluid, imidazole buffer or the glycylglycine damping fluid.
9. according to claim 5 or 6 described creatine diagnosis reagent kits, it is characterized in that: described reduced coenzyme is a kind of in NADPH, NADH, the thio-NADH reduced form nicotinamide coenzyme.
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|---|---|---|---|
| CN 200410064891 CN1757755B (en) | 2004-10-10 | 2004-10-10 | Determination method of creatnine content and reagent box for diagnosing creatnine |
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|---|---|---|---|
| CN 200410064891 CN1757755B (en) | 2004-10-10 | 2004-10-10 | Determination method of creatnine content and reagent box for diagnosing creatnine |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4134793A (en) * | 1975-08-28 | 1979-01-16 | Kyowa Hakko Kogyo Co., Ltd. | Creatinine desimidase in the quantitative determination of creatinine |
| EP0437254B1 (en) * | 1990-01-11 | 1995-12-27 | Roche Diagnostics GmbH | Method and reagent for enzymatic determination of creatinine |
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2004
- 2004-10-10 CN CN 200410064891 patent/CN1757755B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4134793A (en) * | 1975-08-28 | 1979-01-16 | Kyowa Hakko Kogyo Co., Ltd. | Creatinine desimidase in the quantitative determination of creatinine |
| EP0437254B1 (en) * | 1990-01-11 | 1995-12-27 | Roche Diagnostics GmbH | Method and reagent for enzymatic determination of creatinine |
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