CN1731146A - A method for detecting alpha-L-fucosidase vitality and agent therefor - Google Patents
A method for detecting alpha-L-fucosidase vitality and agent therefor Download PDFInfo
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- CN1731146A CN1731146A CN 200510060400 CN200510060400A CN1731146A CN 1731146 A CN1731146 A CN 1731146A CN 200510060400 CN200510060400 CN 200510060400 CN 200510060400 A CN200510060400 A CN 200510060400A CN 1731146 A CN1731146 A CN 1731146A
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Abstract
The invention relates to a method for measuring the slurryalpha-L-fucose glycosidase (AFU) vitality and its agent in the field of biochemistry technology. The invention proportionally adds the tested sample into the agent and mixes them at 30-40 degree about 1.5-10 minutes, then it uses full automation biochemistry analysis to measure the linear changing rate of the absorbance at 340nm to compute the vitality of the sample AFU. The agent for measuring the slurryalpha-L-fucose glycosidase (AFU) vitality comprises a buffer liquid, a preservation, a stabilizer, a bottom object, a surface activator and oxid-type niacinamide cobamaide analogy.
Description
Technical field
The invention relates to the assay method and the reagent of serum alpha-L-fucosidase (AFU) vigor, belong to technological field of biochemistry.
Background technology
Alpha-L-fucosidase (α-L-fucosidase, classification AFU) is called alpha-L-fucosidase fucose hydrolytic enzyme, its catalysis glycosidic bond hydrolysis, participation contains the kalabolism of compound sugar, glycopeptide, glycoprotein and the glycolipid of fucose.Being distributed widely in each tissue such as liver, brain, kidney, pancreas and placenta of human body, being present in the cell, be positioned lysosome, is acid hydrolase, is in the nature glycoprotein.In recent years, a large amount of research datas show, AFU is the reliability index of the mark of primary carcinoma of liver and diagnosis still not, but also can be used for whether the clinical monitoring patient with liver cirrhosis cancerates and the observation index of diabetes state of an illness control.
At present, measure the reagent of alpha-L-fucosidase, all have some shortcomings or weak point.As: powdered reagent need to redissolve and also the redissolution time is long, because of the different bottle of the people differences that redissolve bigger.With PNP is the two-point method liquid reagent of substrate, and the time of assaying reaction is longer; Make the reagent of substrate in addition with CPNPF or CNPF, though reagent need not redissolve, the robotization of sample detects can carry out in batches, but the detection time of such reagent is still longer relatively, and the reaction time reaches 10 minutes; Though the reagent that has is shorter detection time, substrate equally also can spontaneous hydrolysis and turn to be yellow, and influence detects effect.Above-mentioned these known methods all are to adopt the wavelength of 405nm to detect, and will have a strong impact on the detection effect when detecting the turbid sample of high cholerythrin, haemolysis, fat, and testing result even negative can occur is so the value that measure this moment is inaccurate fully.
Summary of the invention
Technical matters to be solved by this invention is the deficiency that overcomes the above-mentioned background technology, the method of a kind of detection alpha-L-fucosidase (AFU) vigor and the improvement of reagent are provided, this method has short, testing result characteristics accurate, simple to operation detection time, and this reagent has that short, highly sensitive, strong interference immunity of reaction time, substrate can not spontaneous hydrolysis take place in the preservation process and the situation of turning to be yellow.
Technical scheme provided by the invention is: a kind of method that detects the alpha-L-fucosidase vigor, test sample is added mixing in the reagent in proportion, hatched 1.5-10 minute at 30-40 ℃, so that the substrate hydrolysis in the AFU catalytic reagent in the sample, generate the L-fucose, the product L-fucose reaction of L-FD and generation, make the reduction of oxidized form nicotinamide coenzyme analog simultaneously, detect the linear change speed of absorbance in the 340nm place with automatic clinical chemistry analyzer then, thereby calculate the vigor of AFU in the sample.
The computing formula of described sample vigor is: AFU activity=(Δ A/min * TV * 1000)/(ε * SV * P).
A kind of reagent that detects the alpha-L-fucosidase vigor, comprise damping fluid, antiseptic, stabilizing agent, substrate, surfactant, also comprise following oxidized form nicotinamide coenzyme analog: any one between oxidized form nicotinamide coenzyme II, oxidized form nicotinamide coenzyme I, sulfo--oxidized form nicotinamide coenzyme I, the sulfo--oxidized form nicotinamide coenzyme II.
Described reagent is single reagent, comprising damping fluid 20-200mmol/L, antiseptic 0.1-5mmol/L, stabilizing agent 0.01-20mmol/L, oxidized form nicotinamide coenzyme analog 0.1-10mmol/L, substrate 0.5-50mmol/L, surfactant 0.1-20ml/L.
Described reagent is double reagent, and wherein reagent A comprises damping fluid 20-150mmol/L, antiseptic 2.5mmol/L-7mmol/L, stabilizing agent 0.01-18mmol/L, substrate 5-18mmol/L, surfactant 2-10ml/L; Reagent B comprises damping fluid 80-100mmol/L, antiseptic 2.5-7mmol/L, stabilizing agent 2-12mmol/L, oxidized form nicotinamide coenzyme analog 6-8mmol/L.
Described damping fluid is: the combination in any of a kind of between trishydroxymethylaminomethane, phosphate, glycocoll, citric acid, borate, HEPES, PIPES, MES, imidazoles, the acetate buffer solution or some kinds, its pH value is 3-9.
Described antiseptic is: the combination in any of a kind of between Sodium azide, thimerosal, P-hydroxybenzoic acid, thiocarbamide, the microbiotic or some kinds.
Described stabilizing agent is: the combination in any of a kind of between disodium EDTA, BSA, magnesium chloride, reduced glutathione, DTT, trehalose, the ethylene glycol or some kinds.
Described substrate is: a kind of between [a-L-(-)-fucose 1-phosphoric acid two (cyclohexylamine) salt] glycosides, a-L-(-)-fucose D-glucoside, a-L-(-)-fucose D-galactoside, a-L-(-)-fucose lactoside, a-L-(-)-fucose D-fucoside.
Described surfactant is: the combination in any of a kind of between GenapolX-080, Tween-20, NP-40, the OP emulsifying agent or some kinds.
Method provided by the invention is because (under the ultraviolet light) detects at the 340nm place, thereby sensitivity is higher, and accuracy rate is higher, and the reaction time is lacked (having only 5-6 minute), and directly detects on automatic biochemistry analyzer, and method of operating is simple and convenient.
Reagent provided by the invention is owing to detect under ultraviolet light, thereby the reaction time is short, highly sensitive; Again because what adopt is the substrate of acomia color group, in the time of 2-8 ℃, can preserve the situation that can not spontaneous hydrolysis take place in a year and turn to be yellow.
Embodiment
Mechanism of the present invention is: the AFU catalytic substrate, generate product L-fucose, product L-fucose reaction by L-FD and generation, make the reduction of oxidized form nicotinamide coenzyme analog simultaneously and make 340nm place absorbance that the rising variation take place, the degree of its rising is directly proportional with AFU activity in the sample, thereby calculates the vigor of AFU in the sample.
Thereby detection method of the present invention mainly is that test sample is added mixing in the reagent in proportion, (incubation temperature and time also can be selected separately to hatch 1.5-10 minute at 30-40 ℃, recommending 37 ℃ hatched 1.5-6 minute), by α in the sample-FU catalytic substrate hydrolysis, generate the L-fucose, product L-fucose by L-FD and generation reacts then, make the reduction of oxidized form nicotinamide coenzyme analog simultaneously and make 340nm place absorbance that the rising variation take place, and with automatic clinical chemistry analyzer in the 340nm place, monitor linear velocity, thereby calculate the vigor of AFU in the sample.
In order to achieve the above object, the designed alpha-L-fucosidase of the present invention is measured reagent, comprises damping fluid, antiseptic, stabilizing agent, oxidized form nicotinamide coenzyme analog, substrate, surfactant.The ratio of each biochemical raw material is determined respectively according to the different of single agents and double reagent.Usually, double reagent can reduce the interference between each biochemical raw material, improves stability.This manufacturing of measuring reagent is comparatively simple, only above-mentioned each biochemical raw material need be mixed in definite ratio.
Described oxidized form nicotinamide coenzyme analog is: a kind of between NADP (oxidized form nicotinamide coenzyme II), NAD (oxidized form nicotinamide coenzyme I), thin-NAD (sulfo--oxidized form nicotinamide coenzyme I), the thin-NADP (sulfo--oxidized form nicotinamide coenzyme II).
Described damping fluid is: trishydroxymethylaminomethane (Tris), phosphate, glycocoll, citric acid, borate, HEPES (N-2-hydroxyethyl piperazine-N-2-ethyl sulfonic acid), PIPES (piperazine-N, two (2-ethanesulfonic acid) sodium salts of N-), the combination in any of a kind of between the MES (2-(N-morphine quinoline) ethyl sulfonic acid), imidazoles, acetate buffer solution or some kinds, its pH value is 3-9.
Described antiseptic is: the combination in any of a kind of between Sodium azide, thimerosal, P-hydroxybenzoic acid, thiocarbamide, the microbiotic or some kinds.
Described stabilizing agent is: the combination in any of a kind of between disodium EDTA, BSA, magnesium chloride, reduced glutathione, DTT, trehalose, the ethylene glycol or some kinds.
Described oxidized form nicotinamide coenzyme analog is: any one between NADP, NAD, thin-NAD, the thin-NADP.
Described substrate is the glycoside compounds of a-L-fucose, can generate the L-fucose behind the AFU catalyzing hydrolysis.Can be: [two (cyclohexylamine) salt of a-L-(-)-fucose 1-phosphoric acid] glycosides { any one between α-L-(-)-Fucose1-phosphate bis (cyclohexylammonium) salt}, a-L-(-)-fucose D-glucoside, a-L-(-)-fucose D-galactoside, a-L-(-)-fucose lactoside, a-L-(-)-fucose D-fucoside.
Described surfactant is: the combination in any of a kind of between GenapolX-080, Tween-20, NP-40, the OP emulsifying agent or some kinds.
The equal buyable of buffering agent of the present invention, antiseptic, stabilizing agent, oxidized form nicotinamide coenzyme analog (being called for short coenzyme in following examples), substrate, surfactant and other biochemical raw material obtains.
Embodiment 1 (detection method):
The basic operation that double reagent is measured:
| Admixture | Blank pipe | Measure pipe |
| Reagent 1 (μ l) | 200 | 200 |
| Distilled water (μ l) | 25 | —— |
| Sample (μ l) | —— | 25 |
| Mixing was hatched 9 minutes for 30 ℃ | ||
| Reagent 2 (μ l) | 50 | 50 |
| Mixing, 30 ℃ are prolonged 60 seconds, and the absorbance in the linear phase of wavelength 340nm place assaying reaction changes, and then calculates the absorbance rate of change Δ A/min. of average per minute | ||
Computing formula:
TV: total reaction volume SV: sample volume
ε: NADH is at the mM extinction coefficient of 340nm: 6.22
P: be the cuvette optical path
Embodiment 2 (detection method):
The basic operation that single reagent is measured:
The basic operation that single reagent is measured:
| Admixture | Blank pipe | Measure pipe |
| Reagent (μ l) | 200 | 200 |
| Distilled water (μ l) | 25 | —— |
| Sample (μ l) | —— | 25 |
| Mixing was hatched 90 seconds for 40 ℃, and the absorbance in the linear phase of wavelength 340nm place assaying reaction changes, and then calculated the absorbance rate of change Δ A/min. of average per minute | ||
Computing formula is with preceding identical.
Embodiment 3 (double reagent):
Reagent A (R1)
Damping fluid MES 20mmol/L
Antiseptic thiocarbamide 2.5mmol/L
Stabilizing agent DTT 0.005mmol/L
Stabilizing agent EDTA.2Na 0.005mmol/L
Surfactant NP-40 3ml/L
Substrate a-L-(-)-fucose-D-glucoside 5mmol/L
Reagent B (R2)
Damping fluid Tris 80mmol/L
Antiseptic Sodium azide 2.5mmol/L
Stabilizing agent trehalose 10mmol/L
Stabilizing agent EDTA.2Na 2mmol/L
Coenzyme NAD P 8mmol/L
Embodiment 4 (double reagent):
Reagent A (R1)
Damping fluid glycocoll 100mmol/L
Antiseptic Sodium azide 5mmol/L
Stabilizing agent EDTA.2Na 2mmol/L
Surfactant Tween-20 2ml/L
Substrate a-L-(-)-fucose-D-galactoside 18mmol/L
Reagent B (R2)
Damping fluid Tris 100mmol/L
Antiseptic Sodium azide 7mmol/L
Stabilizing agent BSA 8mmol/L
Stabilizing agent EDTA.2Na 2mmol/L
Coenzyme NAD P 6mmol/L
Embodiment 5 (double reagent):
Reagent A (R1)
Damping fluid citric acid 150mmol/L
Antiseptic thiocarbamide 7mmol/L
Stabilizing agent ethylene glycol 4mmol/L
Stabilizing agent EDTA.2Na 4mmol/L
Stabilizing agent magnesium chloride 12mmol/L
Surfactant GenapolX-080 10ml/L
Substrate a-L-(-)-fucose-fucoside 15mmol/L
Reagent B (R2)
Damping fluid borate 100mmol/L
Antiseptic Sodium azide 5mmol/L
Stabilizing agent EDTA.2Na 2mmol/L
Coenzyme NAD P 8mmol/L
Embodiment 6 (single reagent):
Damping fluid glycocoll 20mmol/L
Stabilizing agent EDTA.2Na 0.005mmol/L
Stabilizing agent trehalose 0.005mmol/L
Surfactant NP-40 20ml/L
Substrate a-L-(-)-fucose-D-glucoside 0.5mmol/L
Antiseptic Sodium azide 0.1mmol/L
Coenzyme Thin-NADP 0.1mmol/L
Embodiment 7 (single reagent):
Damping fluid MES 200mmol/L
Stabilizing agent EDTA.2Na 2mmol/L
Stabilizing agent ethylene glycol 18mmol/L
Surfactant GenapolX-080 0.1ml/L
Substrate a-L-(-)-fucose-D-glucoside 50mmol/L
Antiseptic Sodium azide 5mmol/L
Coenzyme Thin-NADP 10mmol/L
Embodiment 8 (single reagent):
Damping fluid glycocoll 100mmol/L
Stabilizing agent EDTA.2Na 5mmol/L
Stabilizing agent trehalose 5mmol/L
Surfactant NP-40 10ml/L
Substrate a-L-(-)-fucose-D-glucoside 25mmol/L
Antiseptic Sodium azide 0.1mmol/L
Coenzyme Thin-NADP 50mmol/L.
Claims (10)
1, a kind of method that detects the alpha-L-fucosidase vigor, it is characterized in that test sample is added mixing in the reagent in proportion, 30-40 ℃ hatch 1.5-10 minute after, so that the substrate hydrolysis in the AFU catalytic reagent in the sample, generate the L-fucose, the product L-fucose reaction of L-FD and generation, make the reduction of oxidized form nicotinamide coenzyme analog simultaneously, detect the linear change speed of absorbance in the 340nm place with automatic clinical chemistry analyzer then, thereby calculate the vigor of AFU in the sample.
2, a kind of method that detects the alpha-L-fucosidase vigor according to claim 1 is characterized in that the computing formula of described sample is: AFU activity=(Δ A/min * TV * 1000)/(ε * SV * P).
3, a kind of reagent that detects the alpha-L-fucosidase vigor according to claim 1, it is characterized in that reagent comprises damping fluid, antiseptic, stabilizing agent, substrate, surfactant, also comprise following oxidized form nicotinamide coenzyme analog: any one between oxidized form nicotinamide coenzyme II, oxidized form nicotinamide coenzyme I, sulfo--oxidized form nicotinamide coenzyme I, the sulfo--oxidized form nicotinamide coenzyme II.
4, a kind of reagent that detects the alpha-L-fucosidase vigor according to claim 3, it is characterized in that described reagent is single reagent, comprising damping fluid 20-200mmol/L, antiseptic 0.1-5mmol/L, stabilizing agent 0.01-20mmol/L, oxidized form nicotinamide coenzyme analog 0.1-10mmol/L, substrate 0.5-50mmol/L, surfactant 0.1-20ml/L.
5, a kind of reagent that detects the alpha-L-fucosidase vigor according to claim 3, it is characterized in that described reagent is double reagent, wherein reagent A comprises damping fluid 20-150mmol/L, antiseptic 2.5-7mmol/L, stabilizing agent 0.01-20mmol/L, substrate 5-18mmol/L, surfactant 2-10ml/L; Reagent B comprises damping fluid 80-100mmol/L, antiseptic 2.5-7mmol/L, stabilizing agent 2-12mmol/L, oxidized form nicotinamide coenzyme analog 6-8mmol/L.
6, according to claim 3 or 4 or 5 described a kind of reagent that detect the alpha-L-fucosidase vigor, it is characterized in that described damping fluid is: the combination in any of a kind of between trishydroxymethylaminomethane, phosphate, glycocoll, citric acid, borate, HEPES, PIPES, MES, imidazoles, the acetate buffer solution or some kinds, its pH value is 3-9.
7, according to claim 3 or 4 or 5 described a kind of reagent that detect the alpha-L-fucosidase vigor, it is characterized in that described antiseptic is: the combination in any of a kind of between Sodium azide, thimerosal, P-hydroxybenzoic acid, thiocarbamide, the microbiotic or some kinds.
8, according to claim 3 or 4 or 5 described a kind of reagent that detect the alpha-L-fucosidase vigor, it is characterized in that the stabilizing agent of stating is: the combination in any of a kind of between disodium EDTA, BSA, magnesium chloride, reduced glutathione, DTT, trehalose, the ethylene glycol or some kinds.
9, according to claim 3 or 4 or 5 described a kind of reagent that detect the alpha-L-fucosidase vigor, it is characterized in that described substrate is: the combination in any of a kind of between [a-L-(-)-fucose 1-phosphoric acid two (cyclohexylamine) salt] glycosides, [two (cyclohexylamine) salt of a-L-(-)-fucose 1-phosphoric acid] glycosides, a-L-(-)-fucose D-glucoside, a-L-(-)-fucose D-galactoside, a-L-(-)-fucose lactoside, a-L-(-)-fucose D-fucoside or some kinds.
10, according to claim 3 or 4 or 5 described a kind of reagent that detect the alpha-L-fucosidase vigor, it is characterized in that described surfactant is: the combination in any of a kind of between GenapolX-080, Tween-20, NP-40, the OP emulsifying agent or some kinds.
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| CN102967571A (en) * | 2012-12-24 | 2013-03-13 | 北京利德曼生化股份有限公司 | Liquid single reagent for detecting alpha-L-fucosidase and preparation method thereof |
| CN103025749A (en) * | 2010-06-01 | 2013-04-03 | 格礼卡姆股份公司 | Polymorphs of 2'-o-fucosyllactose and producing thereof |
| CN103063487A (en) * | 2012-12-19 | 2013-04-24 | 中国科学院微生物研究所 | Method for preparing enzyme and substrate compound crystals |
| CN104215631A (en) * | 2014-08-14 | 2014-12-17 | 苏州康铭诚业医用科技有限公司 | Buffer for determining human serum glutamyltransferase |
| CN104267178A (en) * | 2014-10-15 | 2015-01-07 | 宁波美康生物科技股份有限公司 | Serum AFU detection kit |
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| CN103063487A (en) * | 2012-12-19 | 2013-04-24 | 中国科学院微生物研究所 | Method for preparing enzyme and substrate compound crystals |
| CN102967571A (en) * | 2012-12-24 | 2013-03-13 | 北京利德曼生化股份有限公司 | Liquid single reagent for detecting alpha-L-fucosidase and preparation method thereof |
| CN104215631A (en) * | 2014-08-14 | 2014-12-17 | 苏州康铭诚业医用科技有限公司 | Buffer for determining human serum glutamyltransferase |
| CN104267178B (en) * | 2014-10-15 | 2016-01-13 | 宁波美康生物科技股份有限公司 | A kind of serum fucoside enzyme detection kit |
| WO2016058511A1 (en) * | 2014-10-15 | 2016-04-21 | 宁波美康生物科技股份有限公司 | Serum fucosidase detection kit |
| CN104267178A (en) * | 2014-10-15 | 2015-01-07 | 宁波美康生物科技股份有限公司 | Serum AFU detection kit |
| EP3208613A4 (en) * | 2014-10-15 | 2018-03-21 | Ningbo Medical System Biotechnology Co. Ltd. | Serum fucosidase detection kit |
| CN104359846A (en) * | 2014-12-05 | 2015-02-18 | 重庆中元生物技术有限公司 | Kit for detecting content of alpha-L-fucosidase |
| CN104458617A (en) * | 2014-12-05 | 2015-03-25 | 重庆中元生物技术有限公司 | Alpha-hydroxybutyrate dehydrogenase detection kit and preparation method thereof |
| CN107653298A (en) * | 2017-11-15 | 2018-02-02 | 浙江夸克生物科技有限公司 | Adenosine deaminase determines kit |
| CN111088320A (en) * | 2019-12-27 | 2020-05-01 | 桂林英美特生物技术研究所 | α -L-fucosidase determination reagent |
| CN112986164A (en) * | 2021-05-18 | 2021-06-18 | 上海执诚生物科技有限公司 | Anti-heparin stable alpha-L-fucosidase detection kit and application thereof |
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