CN108913752A - A kind of Leucine aminopeptidase detection reagent box haveing excellent performance - Google Patents
A kind of Leucine aminopeptidase detection reagent box haveing excellent performance Download PDFInfo
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- CN108913752A CN108913752A CN201810647159.0A CN201810647159A CN108913752A CN 108913752 A CN108913752 A CN 108913752A CN 201810647159 A CN201810647159 A CN 201810647159A CN 108913752 A CN108913752 A CN 108913752A
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- leucine aminopeptidase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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Abstract
The invention discloses a kind of Leucine aminopeptidase detection reagent box haveing excellent performance, this kit belongs to clinical vitro detection reagent technique field using the leucine aminopeptidase in continuous monitoring method detection blood.Kit of the present invention only includes reagent R.By preferentially having selected B-R buffer into reagent R, it joined suitable polyethylene glycol 20,000, N- beta-hydroxy ethyl-3-acetic acid ethylenediamine, sodium chloride, L- leucyl -2- naphthylamine hydrochloride, Brom-nitro-dioxan (BND), the stability for improving kit in reagent, the range of linearity is preferable, the accuracy of reagent is good, the advantages that at low cost, the present invention are conducive to further promote the use of in the market.
Description
Technical field
The present invention relates to clinical vitro detection reagent technique field, in particular to a kind of leucine amino peptide haveing excellent performance
Enzyme detection kit.
Background technique
Leucine aminopeptidase (leucine aminopeptidase, LAP) is that one kind is widely present in tissue
Proteolytic enzyme, be distributed widely in liver, pancreas, gallbladder, kidney, small intestine and uterus of human body etc. tissue in, especially most with content in liver
Horn of plenty.Leucine aminopeptidase can hydrolyzing N-end be specifically the peptide chain of leucine, rise in various vital movements
Important function.
LAP rich in liver, participates in extremely complex metabolic function, increases when liver cell lesion causes largely to divide
It grows, the enzyme r e leases such as LAP of a large amount of synthesis secretions enter blood and increase its activity, therefore are increased according to enzymatic activity in serum, can
Judge the property and degree of hepatic disease to diagnose the illness.All kinds of hepatopaths are because of hepatocellular injury, and the activity of serum LAP is
There is different degrees of raising.From slight, moderate to severe, serum LAP level increases general chronic hepatitis, can be to a certain degree
It is upper reflection the state of an illness weight, and primary carcinoma of liver LAP increase it is especially pronounced.Therefore, the active detection of serum LAP can be from difference
The generation of various hepatopathys, development are reflected in side.In addition to clinically generally acknowledged A type fetoprotein, alanine aminotransferase, asparagus fern ammonia
Outside the indexs such as sour transaminase, paddy acyl transaminase, in recent years, the active diagnosis and the state of an illness as to all kinds of hepatopathys of serum LAP is estimated
The good Enzyme target of meter is taken seriously.
Currently, the detection method for leucine aminopeptidase mainly has continuous monitoring method, dimethylaminobenzaldehyde colour developing
Method and fluorescence method etc..LAP is not high to the specific requirements of substrate, can hydrolyze a variety of L-Leu derivatives, and serum LAP is living at present
Property measurement mostly use L- leucyl -2- naphthylamines, L- leucyl-p-NA or L- leucyl carboxyanilino be substrate.It is based on
Each substrate of LAP catalyzing hydrolysis generates coloured aryl amine derivatives and carrys out quantitative enzyme, at present L- leucyl-p-NA (Leu-
PNA) the most commonly used as substrate, but the substrate is easy to happen substrate discoloration, will be a greater impact to when being designed to single reagent
Detection is just as a result, and L- leucyl -2- naphthylamines is more stable with respect to other substrates effectively.Therefore, the present invention is with L- leucyl-β-
Naphthylamines is substrate, provides a kind of kit with continuous monitoring method detection leucine aminopeptidase haveing excellent performance, it is anti-to optimize it
System is answered, makes its is reproducible, stability is good, can carry out batch sample full-automation to detect.
Summary of the invention
The object of the present invention is to provide a kind of good examinations for being used to detect leucine aminopeptidase of stability haveing excellent performance
Agent box, the kit use continuous monitoring method.The kit is compared with conventional kit, and stability and the range of linearity are than conventional
Detection kit to be conducive to the popularization and application of reagent clinically well.
Basic principle：
For this kit using L- leucyl -2- naphthylamine hydrochloride as substrate, leucine aminopeptidase, which is decomposed, releases Huang
The 2- naphthylamines of color detects absorbance change rate at dominant wavelength 450nm, a length of 660nm of complementary wave, measures leucine aminopeptidase
Activity value.
What invention was obtained through the following steps：
A kind of Leucine aminopeptidase detection reagent box haveing excellent performance, it is characterised in that include reagent R.Its reagent composition
For：B-R buffer, 20g/L polyethylene glycol 2 ten thousand, 10mmol/L N- beta-hydroxy ethylethylenediamine three of the 40mmol/LpH for 8.6
Acetic acid, 50mmol/L sodium chloride, 4.8mmol/LL- leucyl -2- naphthylamine hydrochloride, 10mmol/LBrom-nitro-dioxan
(BND)。
The Leucine aminopeptidase detection reagent box, it is characterised in that reagent buffer is 25 DEG C, the B- that pH is 8.6
R buffer.
The Leucine aminopeptidase detection reagent box, it is characterised in that the surfactant is polyethylene glycol 20,000.
The Leucine aminopeptidase detection reagent box, it is characterised in that the stabilizer is N- beta-hydroxy ethyl second two
Amine triacetic acid.
The Leucine aminopeptidase detection reagent box, which is characterized in that using automatic clinical chemistry analyzer using speed
Rate method is measured, and detection dominant wavelength is 450nm, a length of 660nm of complementary wave.
Kit of the invention carries out on the automatic clinical chemistry analyzer with double reagent function, specifically used method
It is as follows：
Physiological saline, 10 μ l of sample or calibration object is added, adds the reagent R of 300 μ l, reads extinction after preincubate 3min
A1 is spent, then reads absorbance A 2 after reacting 2min, and calculate Δ A/min.
Beneficial effects of the present invention：
1) preferred buffer of the B-R buffer as reaction system of the present invention is conducive to substrate in raising reaction system
Stability and enzyme reaction activity；
2) heavy metal ion and general heavy metal ion chelating agent all can generate shadow to the activity of leucine aminopeptidase
It rings, therefore the present invention has preferentially selected comparatively gentle stabilizer N- beta-hydroxy ethyl-3-acetic acid ethylenediamine, to reduce heavy metal
The active inhibition of ions enzyme；
3) preferred activator of the polyethylene glycol 40,000 as enzyme reaction system, increases suitable sodium chloride as ionic equilibrium
Agent.
4) the preferred Brom-nitro-dioxan (BND) of the present invention is used as preservative, can both reduce preservative to enzyme activity
The inhibition of property, and can efficiently prevent the raw bacterium of reagent.
Detailed description of the invention
Fig. 1 is the correlation curve figure of two kinds of reagents,
Fig. 2 is that two kinds of reagents imitate phase stability curve figure.
Specific embodiment
Invention is further explained combined with specific embodiments below：
Embodiment 1
A kind of conventional Leucine aminopeptidase detection reagent box haveing excellent performance, reagent set become：
Reagent R described in the present embodiment, when configuration, need to first prepare buffer, after being transferred to proper pH value, then add other materials.
When in use, measuring method is to step auspicious 800 full-automatic biochemical using with double reagent function to kit described in the present embodiment
Analyzer is measured using performance rate method, is operated as follows：
Physiological saline, 10 μ l of sample or calibration object is added, adds the reagent R of 300 μ l, reads extinction after preincubate 3min
A1 is spent, then reads absorbance A 2 after reacting 2min, and calculate Δ A/min.
Δ A/min=(A2-A1)/reaction time
Leucine aminopeptidase content (U/L)=Δ A/min × theory factor
Embodiment 2
Accuracy validation test：Using the Leucine aminopeptidase detection reagent of embodiment 1 as experimental group, obtain in the market
The Leucine aminopeptidase detection reagent that a kind of accuracy haveing excellent performance of approval is good, stability is good is examined as a control group
It surveys, 20 clinical serum samples is detected, testing result is as shown in table 1.Obtain the correlation curve of two kinds of reagents (such as
Shown in Fig. 1), the results showed that, the related coefficient of two group reagent boxes is 0.9989, illustrates that the two correlation is relatively good.Prove this hair
The component of bright kit addition and change will not impact its accuracy, and kit still keeps preferable accuracy.
The Leucine aminopeptidase detection reagent contrasting detection result that 1 embodiment of table, 1 reagent and market are approved
Embodiment 3
Linear dependence verification test：The high level sample that leucine aminopeptidase content is 240U/L is chosen, physiology salt is used
Water is serially diluted, and prepares the sample of 6 various concentrations, concentration be followed successively by 240U/L, 192U/L, 144U/L, 96U/L,
48U/L,0U/L.It is utilized respectively 1 reagent of embodiment and control group reagent is detected, the sample of each concentration measures three respectively
It is secondary, it is averaged respectively, testing result is as shown in table 2.
2 embodiment of table, 1 reagent linear correlation confirmatory experiment testing result
As shown above, embodiment 1 is all larger than 0.990 with group reagent testing result related coefficient is compareed, and embodiment 1 is tried
For the related coefficient of agent testing result slightly larger than the related coefficient of control group reagent testing result, this shows that reagent of the present invention has more
Good linear dependence.
Embodiment 4
Stability confirmatory experiment：The store reagents in 2 DEG C~8 DEG C, the light protected environment of non-corrosive gas detect embodiment
1 and compare group reagent stability.Monthly No. 1 measures same pooled serum sample with two group reagents respectively, and measurement is made even three times
Mean value, detection data are as shown in table 3.
3 embodiment of table, 1 reagent stability confirmatory experiment testing result
Experimental result shows, 1 reagent of embodiment is stored 15 months in 2 DEG C~8 DEG C, the light protected environment of non-corrosive gas
Stablize, and compares group reagent and store 12 months unstable, explanations of beginning in 2 DEG C~8 DEG C, the light protected environment of non-corrosive gas
Reagent stability of the present invention is better than control experiment group.
In conclusion therefore, Leucine aminopeptidase detection reagent box provided by the invention is with good performance advantageous
It is promoted the use of in further in the market.
Claims (6)
1. a kind of Leucine aminopeptidase detection reagent box haveing excellent performance haveing excellent performance includes reagent R, it is characterised in that institute
It is as follows to state reagent composition：
2. Leucine aminopeptidase detection reagent box according to claim 1, it is characterised in that reagent B-R buffer is phosphorus
Sour disodium hydrogen, acetic acid, the concentration of three kinds of substances of sodium tetraborate are the buffering that PH is 8.6 at 25 DEG C of the composition of 40mmol/L
Liquid.
3. Leucine aminopeptidase detection reagent box according to claim 1, it is characterised in that the surfactant is
Polyethylene glycol 20,000.
4. Leucine aminopeptidase detection reagent box according to claim 1, it is characterised in that the stabilizer is N- β-
Oxyethylethylenediaminetriacetic acid.
5. Leucine aminopeptidase detection reagent box according to claim 1, it is characterised in that the preservative is Brom-
Nitro-dioxan (BND) (Roche biology).
6. Leucine aminopeptidase detection reagent box according to claim 1, which is characterized in that use full-automatic biochemical point
Analyzer is measured using performance rate method, and detection dominant wavelength is 450nm, a length of 660nm of complementary wave.
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| CN201810647159.0A CN108913752A (en) | 2018-06-22 | 2018-06-22 | A kind of Leucine aminopeptidase detection reagent box haveing excellent performance |
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| CN201810647159.0A CN108913752A (en) | 2018-06-22 | 2018-06-22 | A kind of Leucine aminopeptidase detection reagent box haveing excellent performance |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864206A (en) * | 2012-09-11 | 2013-01-09 | 宁波美康生物科技股份有限公司 | Anti-heparin interference leucine aminopeptidase measuring reagent |
| CN103266164A (en) * | 2013-05-24 | 2013-08-28 | 宁波美康生物科技股份有限公司 | Leucine aminopeptidase detection reagent |
| CN105838775A (en) * | 2016-04-28 | 2016-08-10 | 安徽伊普诺康生物技术股份有限公司 | Kit for detecting leucyl aminopeptidase and preparation method of kit |
-
2018
- 2018-06-22 CN CN201810647159.0A patent/CN108913752A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864206A (en) * | 2012-09-11 | 2013-01-09 | 宁波美康生物科技股份有限公司 | Anti-heparin interference leucine aminopeptidase measuring reagent |
| CN103266164A (en) * | 2013-05-24 | 2013-08-28 | 宁波美康生物科技股份有限公司 | Leucine aminopeptidase detection reagent |
| CN105838775A (en) * | 2016-04-28 | 2016-08-10 | 安徽伊普诺康生物技术股份有限公司 | Kit for detecting leucyl aminopeptidase and preparation method of kit |
Non-Patent Citations (2)
| Title |
|---|
| MAGDA PYRZYNA等: "Purification,biochemical characterisation,and mass spectrometry analysis of phenylalanine aminopeptidase from the shoots of pea plants", 《ACTA PHYSIOL PLANT》 * |
| 王继贵: "《临床生化检验（第二版）》", 31 July 1996 * |
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Application publication date: 20181130 |