CN109112181A - A kind of 5`- activity of 5 '-nucleotidase measuring method and 5`- nucleotidase detection kit - Google Patents
A kind of 5`- activity of 5 '-nucleotidase measuring method and 5`- nucleotidase detection kit Download PDFInfo
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- CN109112181A CN109112181A CN201710489361.0A CN201710489361A CN109112181A CN 109112181 A CN109112181 A CN 109112181A CN 201710489361 A CN201710489361 A CN 201710489361A CN 109112181 A CN109112181 A CN 109112181A
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Abstract
The present invention relates to a kind of active methods of measurement 5'-nucleotidase, while the invention further relates to 5'-nucleotidase detection kits, belong to medical test determination techniques field.This method is using 5- inosine monophosphate as substrate, using purine nucleoside phosphorylase, xanthine dehydrogenase as toolenzyme, make substrate conversion and dehydrogenation, restores oxidized coenzyme and generate reduced coenzyme, reduced coenzyme production quantity is detected, at wavelength 340nm to calculate enzyme reaction speed.Compared with modern technologies, reaction step of the present invention is few, few using raw material type, and the effect of alkali resistant acid phosphatase is strong, and reagent performance is stablized, and the range of linearity is wide, high sensitivity, and accuracy is good.
Description
Technical field
The present invention relates to a kind of active methods of measurement 5'-nucleotidase, while the invention further relates to 5'-nucleotidases
Detection kit belongs to medical test determination techniques field.
Background technique
5'-NT is the phosphohydrolase of one group with alkaline phosphatase difference biochemical property, be distributed mainly on the heart,
Liver, brain, muscle, kidney and lung etc. are distributed in cholangiole, sinus hepaticus and Kupffer's cells in liver, the content highest in liver cytoplasm,
By liver metabolism.Active raising is mainly seen in hepatobiliary system disease, liver cancer, hepatitis and liver to 5'-NT in blood
Hardening, especially liver cancer are often examined with alpha-fetoprotein, carcinomebryonic antigen and Alpha-Fucosidase and other liver function projects simultaneously
It surveys, comprehensive analysis is carried out to disease in the liver and gallbladder.
Since the measurement of serum 5'-NT has preferable specificity and sensitivity to the diagnosis of liver diseases, nearly 20
Nian Zhong, measuring method are continued to develop and are improved, and the detection reagent of five generation method sums has successively been developed, and main feature can letter
It states as follows:
The first generation: 5- inosine monophosphate deamination is generated trophicardyl and phosphoric acid by 5'-NT.5- flesh is measured by dynamic
Absorbance at one phosphoric acid 265nm of glycosides, can calculate 5'-NT vigor size.Since high concentration of substrate causes absorbance dense
Height is spent, which is only applicable to 5- inosine monophosphate concentration lower than 15umol.Substrate saturation is not achieved in so low concentration of substrate
It is required that 5'-NT Activity determination is caused to be distorted.Therefore, which is not suitable for clinical application.
The second generation: 5- inosine monophosphate deamination is generated trophicardyl and phosphoric acid by 5'-NT, and the latter and ammonium molybdate act on
Phosphomolybdic acid is generated, then is reduced agent and is reduced into molybdenum blue, shade is directly proportional to the phosphate radical of release.Survey phosphorus colorimetric method reagent kind
Class is more, cumbersome, time-consuming, is not suitable for automated analysis, and endogenous phosphoric acid also has interference, and serum neutral and alkali to the measurement
Phosphatase can also hydrolyze 5- inosine monophosphate and generate identical product, need that Ni is added in reaction process2+Ion inhibits alkaline phosphatase
Otherwise the nonspecific reaction of enzyme will affect the accuracy of measurement result.
The third generation: KalckerShi application 5'-NT is coupled purine nucleoside phosphorylase and xanthine oxidase reaction
Continuous detection method.The speed risen by lithate absorbance at measurement 293nm is active to calculate 5'-NT.But
Sera absorbance is too high when 293nm, causes clinical application inconvenient.
Forth generation: being coupled purine nucleoside phosphorylase and xanthine oxidase by 5'-NT, catalase and
Aldehyde dehydrogenase calculates 5 '-nucleosides by the rate that reduced Coenzyme II absorbance at hydroperoxidation measurement 334nm rises
Phytase activity.The method overcomes the difficulty of previous 5'-NT test, however, more in view of reaction step, reagent cost is high,
Hinder practical application.
In 5th generation: 5'-NT is through Mg2+Or Mn2+Time 5- inosine monophosphate is digested after activation generates trophicardyl；Secondary Huang
Glycosides passes through the effect of purine nucleoside phosphorylase again, with PO4 3—Generate hypoxanthine；The latter is raw under xanthine oxidase oxidation
At uric acid and hydrogen peroxide；Last peroxidase decomposes water and generates elemental oxygen, aoxidizes phenyl amines color developing agent and 4- amino peace is replaced
It is reacted than woods, generates the coloured quinone of aubergine.The speed that justice degree rises is inhaled at 550nm to calculate the activity of 5'-NT.
Reaction has strong antijamming capability.The characteristics of being suitble to automation quickly to measure is 5 '-nucleotide of routine clinical development
Enzyme detection reagent creates favorable conditions.This method substantially currently on the market.But the 5th generation method reaction step
More, reaction speed is slow, more using raw material type, many shortcomings such as at high cost.
Summary of the invention
The technical problem to be solved by the present invention is in view of the deficiencies of the prior art, propose that one kind can overcome the above existing skill
The active measuring method of the 5'-NT of art disadvantage, while providing the 5'-NT detection reagent to realize this method
Box.Reaction step is few, few using raw material type, and reagent stability is good, and the good range of linearity is wide, and high sensitivity, accuracy is good, is convenient for
It promotes.
It is as follows that the present invention measures the active method and step of 5'-NT:
1), by sample and mainly by 5 '-inosine monophosphates, phosphate, purine nucleoside phosphorylase, oxidized coenzyme, Huang
The reagent mixing of purine dehydrogenase composition, is allowed to occur to react as follows:
2), detection end reaction object rises change rate in dominant wavelength 340nm absorbance, calculates the work of 5'-nucleotidase
Property size, colored intensity are directly proportional to 5'-nucleotidase activity.
This method has lacked single step reaction compared with the 5th generation method, so that reaction speed is accelerated, xanthine oxidase
It has changed xanthine dehydrogenase into, has removed two color developing agents of 4-AA and phenyl amines color developing agent, using oxidized coenzyme,
Hypoxanthine dehydrogenation carrys out reduction-oxidation type coenzyme and generates reduced coenzyme, detects to reduced coenzyme, eliminates peroxidating
Object enzyme, 4-AA and the color developing agent of phenyl amines color developing agent, especially two are easy by air and water under general condition
Oxygen slowly aoxidize, keep later period blank slowly higher, reaction sensitivity caused to decline.It makes in this reagent of oxidized coenzyme aobvious
Toner, stability is good, and reagent is colorless and transparent, and after reaction generates reduced coenzyme, the change that absorbance rises is detected at 340nmnm
Rate, to calculate the vigor of 5'-NT.Reaction front and back is not by the interference of any factor.
To realize the adenosine deaminase detection kit of the method for the present invention,
Including reagent one:
Reagent two:
By the composition of reagent, reagent one: two=1~4 ︰ 1 of reagent different concentration ratios, preferred reagent one: reagent can be made into
Two=1~3 ︰ 1.Sample: reagent=1: 10~100, preferably 1 ︰ 25~50.
Buffer is the combination of one or both of trishydroxymethylaminomethane, tartaric acid, imidazoles, acetate, phosphate.
Dosage is preferably 50~100mmol/L.
Preservative be sodium azide, ProClin-300, phenolic hydroxyl group class, the oil in benzoic acids from, amount ranges 2~
20mmol/L.General preservative select will according to preservative whether to 5'-NT and toolenzyme whether there is or not depending on inhibiting effect,
Tests prove that above several anti-corrosions can be used in this formula, still, be with sodium azide it is preferred, do not have to reaction system
There is any influence, price is also low, and optimum amount is in 10~15mmol/L.
Stabilizer is Bovine serum albumin, amino acids, carbohydrate, and polyalcohols etc. can be applied alone, can also be two kinds or two
Kind or more combination, preferred Bovine serum albumin, use scope is in 0.5~20g/L.Mannitol can be selected in carbohydrate, and trehalose is polynary
The optional spent glycol of alcohol, PEG6000 etc..
Mn2+、Mg2+The activator of 5'-NT, very sensitive, dosage should be grasped strictly, be added in 0.5~5mmol/L
It can with H2PO4 -Muddy in conjunction with generating, reaction is parabolically；Add that have lacked reaction sensitivity poor.
It can be suitably added surfactant in reaction system, reaction can be accelerated, increase the stability of enzyme, including Tweens,
The nonionic surfactants such as polyethenoxy ether class, particularly preferably Tweens, use scope is in 0.5~5.0ml/L, most preferably
Select 1.0~1.5ml/L.
Color developing agent, it is preferable to use oxidized coenzyme I, cheap, is used using oxidized coenzyme I or oxidized coenzyme Ⅱ
For range in 0.5~10.0g/L, actual amount should be depending on ratio of reagents.
The technology of 5'-NT detection reagent key is: 1, how rapidly to inhibit the activity of alkaline phosphatase, prevent
Alkaline phosphatase decomposes rapidly substrate 5 '-inosine monophosphate, and 2, using suitable 5'-NT metal activator, and gold
Belong to activator to get along well again H2PO4 —Generate turbidity and precipitation.Pervious method is to use sodium β-glycerophosphate, unexpectedly the inhibition alkalinity phosphorus of striving property
The activity of sour enzyme prevents cross reaction, but dosage is at high cost greatly, and inhibitory effect is not very good.It is suitable that this formula uses
Buffer, is both buffer and Inhibitors of Alkaline Phosphatase, and inhibitory effect is fine.In addition Mn is selected2+And H2PO4 —Suitably
Concentration proportioning will not generate muddy and precipitating.
Specific embodiment
Embodiment one
Reagent one:
Reagent two:
Reagent one is 2 ︰ 1, performance rate method, positive reaction, wavelength selection 340nm, sample, reagent one and reagent than reagent two
Two ratio is 8 ︰, 180 ︰ 90, and 37 DEG C of temperature, serum reacts 5 minutes with reagent one, and reagent two is added, delay time 2 minutes, detects
2 minutes.
Embodiment two
Reagent one:
Reagent two:
The amount ratio of reagent one and reagent two is 4 ︰ 1, performance rate method, positive reaction, wavelength selection 340nm, sample, reagent one
Volume ratio with reagent two is 6 ︰, 200 ︰ 50, and 37 DEG C of temperature, serum reacts 5 minutes with reagent one, and reagent two, delay time is added
It 2 minutes, detects 2 minutes.
Embodiment three
Reagent one:
Reagent two
The amount ratio of reagent one and reagent two is 3 ︰ 1, performance rate method, positive reaction, wavelength selection 340nm, sample, reagent one
Volume ratio with reagent two is 8 ︰, 180 ︰ 60, and 37 DEG C of temperature, serum reacts 5 minutes with reagent one, and reagent two, delay time is added
It 2 minutes, detects 2 minutes.
Commercially available 5'-NT is detected using the detection kit, result is
| Theoretical value | 0 | 42.5 | 85 | 127.5 | 170 | 221.5 | 255 | 279.5 | 340nm |
| Measured value | 0.42 | 44.2 | 92.8 | 137.7 | 181.6 | 219.6 | 253.1 | 290.2 | 328.7 |
300U/L or more can linearly be reached.
It uses the same clinical serum as sample, repeats detection 20 times, as a result as follows:
SD=0.8256, CV=1.0812%
50 parts of clinical serums, which are detected, with the detection kit and xanthine dehydrogenase method does comparative test, comparative test result
It is as follows:
The comparative test of 50 clinical samples
The resulting result of two methods is highly relevant, r2=0.9989.
Detection of Stability: it is calibrated with the freeze-drying calibration object of the same batch, to detect with a batch of freeze-drying quality-control product, often
Moon detection is primary, and 13 totally months.One bottle is redissolved every time, is repeated 10 times, is averaged, as a result as follows:
Quality-control product definite value: 45 ± 6U/L
According to national regulation, reagent validity period that enterprise formulates should exceed the time limit still stable in one month, as a result meet the requirements.
The above is only a preferred embodiment of the present invention, protection scope of the present invention is not limited merely to above-mentioned implementation
Example, all technical solutions belonged under thinking of the present invention all belong to the scope of protection of the present invention.It should be pointed out that for the art
Those of ordinary skill for, several improvements and modifications without departing from the principles of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of active method of measurement 5'-nucleotidase, steps are as follows:
1), by sample and mainly by 5 '-inosine monophosphates, phosphate, purine nucleoside phosphorylase, oxidized coenzyme, xanthine
The reagent mixing of dehydrogenase composition, is allowed to occur to react as follows:
2), the speed that detection end reaction object rises in dominant wavelength 340nm absorbance, the activity for calculating 5'-nucleotidase are big
It is small.
2. measuring the active method of 5'-nucleotidase according to claim 1, it is characterised in that: sample and reagent
Ratio is controlled 1/10 to 1/100.
3. a kind of 5'-nucleotidase detection kit, it is characterised in that: including reagent one and reagent two,
Reagent one includes
Reagent two includes
The ratio of the reagent one and reagent two is 1/1 to 4/1.
4. 5'-nucleotidase detection kit according to claim 3, it is characterised in that: the buffer is trihydroxy methyl
One of aminomethane, tartaric acid, imidazoles, acetate, phosphate.
5. 5'-nucleotidase detection kit according to claim 3, it is characterised in that: the pH of buffer be 7.0~
8.0。
6. 5'-nucleotidase detection kit according to claim 3, it is characterised in that: the preservative is Azide
Sodium, ProClin-300, phenolic hydroxyl-compounds, one of benzoic acid compounds.
7. 5'-nucleotidase detection kit according to claim 3, it is characterised in that: the stabilizer is calf serum
Albumin, amino acid, at least one of carbohydrate derivative, polyalcohol.
8. 5'-nucleotidase detection kit according to claim 3, it is characterised in that: the surfactant is
Tweens or polyethenoxy ether class nonionic surfactant.
9. 5'-nucleotidase detection kit according to claim 3, it is characterised in that: the zymoexciter is Mn2+Or
Mg2+, dosage is 1.0~2.5mmol/L.
10. 5'-nucleotidase detection kit according to claim 3, it is characterised in that: the color developing agent is oxidized form
Cozymase or oxidized coenzyme Ⅱ, the dosage of the color developing agent are 0.5~3.0g/L.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710489361.0A CN109112181A (en) | 2017-06-24 | 2017-06-24 | A kind of 5`- activity of 5 '-nucleotidase measuring method and 5`- nucleotidase detection kit |
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| CN201710489361.0A CN109112181A (en) | 2017-06-24 | 2017-06-24 | A kind of 5`- activity of 5 '-nucleotidase measuring method and 5`- nucleotidase detection kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111139284A (en) * | 2020-01-16 | 2020-05-12 | 浙江夸克生物科技有限公司 | High-accuracy 5' -nucleotidase determination kit |
| CN111808918A (en) * | 2020-07-26 | 2020-10-23 | 武汉景川诊断技术股份有限公司 | Kit for determining 5' -nucleotidase |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1749411A (en) * | 2004-09-14 | 2006-03-22 | 王尔中 | Method for measuring 5'-nucleotidase activity and diagnostic kit of 5'-nucleotidase |
| CN1778949A (en) * | 2004-11-23 | 2006-05-31 | 苏州艾杰生物科技有限公司 | Determination of 5'-nucleotidase activity and its diagnostic reagent kit of 5'-nucleotidase |
-
2017
- 2017-06-24 CN CN201710489361.0A patent/CN109112181A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1749411A (en) * | 2004-09-14 | 2006-03-22 | 王尔中 | Method for measuring 5'-nucleotidase activity and diagnostic kit of 5'-nucleotidase |
| CN1778949A (en) * | 2004-11-23 | 2006-05-31 | 苏州艾杰生物科技有限公司 | Determination of 5'-nucleotidase activity and its diagnostic reagent kit of 5'-nucleotidase |
Non-Patent Citations (1)
| Title |
|---|
| BOGAN, KL等: "5 "-Nucleotidases and their new roles in NAD(+) and phosphate metabolism", 《NEW JOURNAL OF CHEMISTRY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN111139284A (en) * | 2020-01-16 | 2020-05-12 | 浙江夸克生物科技有限公司 | High-accuracy 5' -nucleotidase determination kit |
| CN111808918A (en) * | 2020-07-26 | 2020-10-23 | 武汉景川诊断技术股份有限公司 | Kit for determining 5' -nucleotidase |
| CN111808918B (en) * | 2020-07-26 | 2024-01-23 | 武汉景川诊断技术股份有限公司 | Kit for determining 5' -nucleotidase |
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