CN109576343A - A kind of formula of continuity method measurement activity of acid phosphatase kit - Google Patents
A kind of formula of continuity method measurement activity of acid phosphatase kit Download PDFInfo
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- CN109576343A CN109576343A CN201811636997.4A CN201811636997A CN109576343A CN 109576343 A CN109576343 A CN 109576343A CN 201811636997 A CN201811636997 A CN 201811636997A CN 109576343 A CN109576343 A CN 109576343A
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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
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
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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
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/30—Naphthol derivatives, e.g. alpha-naphthyl-esters, i.e. alpha-NE, beta-naphthyl-esters, i.e. beta-NE
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
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Abstract
A kind of formula of continuity method measurement activity of acid phosphatase kit, it is characterized by: being substrate with 4- nitro -1- naphthyl phosphate (4NNPP), it is dissolved in pH and is no more than 5.0 mM between 4.5 ~ 6.0 in 0.05 ~ 0.20 M citric acid-sodium citrate, this is substrate solution, that is, reagent I；Setting reaction total volume is required according to amount of liquid in photometer contrastive colours cup when use, first by reagent I constant temperature to the selected temperature for being no more than 37 degree, set reaction total volume reagent I is taken to be added in cuvette again, 5 ~ 100 ul of sample-adding product, certain absorbing at wavelengths changes between 440 ~ 470 nm are continuously tracked after mixing；Product formation speed is conversed with substrate 4NNPP and its product difference extinction coefficient, indicates activity of acid phosphatase.
Description
Technical field
The invention belongs to field of biotechnology, it is related to serum, animal tissue's homogenate, plant tissue homogenate, animals and plants and micro-
Biological cell lysate is sample, and reaction process is continuously tracked in photometry, quantitative determines contained activity of acid phosphatase in sample
Method correspond to agent prescription.
Background technique
Acid phosphatase refers to the non-specific phosphomonoesterase that optimal pH is lower than 6.0, can hydrolyze the corresponding mono phosphoric acid ester of colour developing group
Ester.Activity of acid phosphatase from samples such as biological tissue, cells is reflection cells growth activity, reproductive physiology ability etc.
Important information.In biological medicine laboratory and Clinical Test Lab, most popular equipment is the spectrophotometric that measurement absorbs
Meter.The measurement most suitable method of enzymatic activity is that enzyme reaction process is continuously tracked to determine that product generates initial velocity and indicates enzymatic activity, this
Method is also referred to as continuity method.So it is the measurement optimal side of acid phosphatase that product, which is continuously tracked, to absorb increase with chromogenic substrate
Method is also easiest to realize automated analysis.
Up to now, this kind of activity of acid phosphatase of reaction process measurement is continuously tracked only to report with the chloro- 4- second of 2,6- bis-
Phenyl phosphate is substrate, and product absorption peak is near 320 nm of ultra-violet (UV) band, and the sample from biological tissue
Usually containing has the substance absorbed by force in ultra-violet (UV) band, interferes to measurement.In order to avoid this interference, with color product relative to
The chromogenic substrate that the maximum poor absorption peak of chromogenic substrate is located at visible region is continuously tracked acid phosphatase reaction process and measures its work
Property is just very necessary.4- nitrophenyl phosphate is the common chromogenic substrate for measuring non-specific phosphomonoesterase, product 4- nitre
Base phenol in alkalinity maximum absorption band near 405nm, and many filter type analytical equipments be provided with measurement 405,
450,610 nm optical filter.But 4- nitrophenol pH be lower than 6.0 when unionization and it is very low in 405 nm extinction coefficients, make
It is too low at continuity method sensitivity.Fortunately, 4- nitro -1- naphthyl phosphate (4NNPP) be chromogenic substrate when, pH 5.0 its
Color product 4- nitro -1- naphthols with respect to chromogenic substrate the poor absorption peak wavelength of maximum in 455 ~ 458nm, in pH 5.0 450
6.0 (mM) are still greater than in nm mMs of extinction coefficient-1·cm-1, therefore the chromogenic substrate absorbs continuously suitable for tracking product
Method measures activity of acid phosphatase；Using shorter than 440 nm or it is longer than 470 nm wavelength and is also applied for being continuously tracked product and absorbs and become
Change, but sensitivity can reduce.
For convenient for application, the present invention selects this chromogenic substrate to measure acid phosphatase, and is further configured to kit.This
It is chromogenic substrate that the core content of invention protection, which is with 4- nitro -1- naphthyl phosphate, is directly dissolved in required citric acid -
It in sodium citrate buffer solution, can be used to measure using only a kind of reagent, particularly convenient for using on automatic clinical chemistry analyzer；Make
With other than citric acid-sodium citrate other buffers and those skilled in the art known in interchangeable content, also wrap
It is contained in claim of the invention.
Summary of the invention
The present invention describes a kind of formula of continuity method measurement activity of acid phosphatase kit, is primarily characterized in that:
It is chromogenic substrate with 4- nitro -1- naphthyl phosphate, is dissolved in pH 0.05 ~ 0.20 M citric acid-lemon between 4.5 ~ 6.0
In sour sodium, for concentration between 0.10 mM to 5.0 mM, adding Sodium azide, benzoic acid or antibiotic is preservative, is as substrate solution
Reagent I；
PH 0.05 ~ 0.20 M citric acid-sodium citrate between 4.5 ~ 6.0, adding Sodium azide, benzoic acid or antibiotic is that preservative is
Reagent II is used for dilute sample and substrate solution；Substrate solution is diluting constant temperature again with reagent II using preceding, and sample is used before facing measurement
Reagent II dilution；
Further, setting reaction total volume is required according to amount of liquid in photometer contrastive colours cup when use；First by reagent I constant temperature to not
More than 37 degree, then set reaction total volume reagent I is taken to be added in cuvette, is loaded 2 ~ 100 ul of product, is continuously tracked after mixing
Certain absorbing at wavelengths changes between 440 ~ 470 nm；Product formation speed is conversed with substrate 4NNPP and its product difference extinction coefficient,
Indicate activity of acid phosphatase.
Further, the formula of a kind of continuity method measurement activity of acid phosphatase kit, feature also:
Sample is such as not required to dilute, substrate solution I is not also diluted, then only needs reagent I without reagent II；
Physiological saline also is suitable as reagent II for dilute sample；
In reagent I, the inhibitor for inhibiting specific Acid Phosphatase Isozymes is added, realizes the selectivity to specific acid phosphatase
Measurement；
In reagent I, in addition to citric acid-sodium citrate buffer solution, other buffers that can cover pH 5.5 are also suitable.
Detailed description of the invention
Fig. 1 acid phosphatase enzyme hydrolysis 4NNPP reaction process；Buffer zeroing, Change of absorption of 300 nm to 600 nm
Fig. 2 acid phosphatase enzyme hydrolysis 4NNPP reaction process；4NNPP zeroing, 400 nm to 500 nm Change of absorption
Fig. 3 acid phosphatase enzyme hydrolysis 4NNPP reaction process；440,457,465 nm absorb the variation with the reaction time
Fig. 4 measures 4- nitro -1- naphthols in the extinction coefficient of 5.5 wavelength 460 of pH
The Michaelis constant of Fig. 5 measurement acid phosphatase enzyme hydrolysis 4NNPP
The optimal pH of Fig. 6 measurement acid phosphatase enzyme hydrolysis 4NNPP.
Specific embodiment
Agents useful for same and solution are as follows in embodiment:
4NNPP synthesis, details are shown in: " Medical University Of Chongqing's journal ", volume 2013,38, the 04th phase, pp 413-416.
0.2 M citric acid-sodium citrate buffer solution, pH 4.5 ~ 6.0
Reagent II:0.20 M citric acid-sodium citrate, pH 5.5
Reagent I:4NNPP is dissolved in reagent II, 1.0 mM
Instrument: 2000 spectrophotometer of shimadzu
4- nitro -1- naphthols: it is purchased from Alfa-Aesar
Pig refining is derived from farm nearby.
Embodiment 1, acid phosphatase enzyme hydrolysis 4NNP reaction process；Buffer zeroing,
Pig refining reagent II is diluted 100 times for sample after dilution；Sample after 20 μ l dilute is added in 2.0 ml reagent I
Product, vortex are mixed, are immediately transferred in 4.0 ml cuvettes, and scanning absorption spectrum changes with time, as a result such as Fig. 1.It reduces
Absorption spectrum variation such as Fig. 2 after 4NNPP absorbs in reaction mixture.The corresponding absorption at 440,457,465nm is at any time
Fig. 3 is shown in variation
Embodiment 2, measurement product extinction coefficient, Michaelis constant, optimal pH
4- nitro -1- naphthols is dissolved in reagent II, 460 nm of measurement absorb the variation with concentration, as a result such as Fig. 4.
Reagent I is diluted with reagent II, obtains the substrate solution of various concentration；Measure 100 times dilution after sample activity；Double inverses
Mapping, obtains Km close to 0.9 mM, sees Fig. 5.
With the different molten 4NNPP of pH buffer to 1.0 mM, 460 nm Change of absorption initial velocity are surveyed；Activity is shown in figure with pH variation
6。
Claims (2)
1. a kind of formula of continuity method measurement activity of acid phosphatase kit, is characterized in that:
(1) it is chromogenic substrate with 4- nitro -1- naphthyl phosphate, is dissolved in pH 0.05 ~ 0.20 M citric acid-between 4.5 ~ 6.0
In sodium citrate, for concentration between 0.10 mM to 5.0 mM, adding Sodium azide, benzoic acid or antibiotic is preservative；This is substrate
Liquid, that is, reagent I；
(2) pH 0.05 ~ 0.20 M citric acid-sodium citrate between 4.5 ~ 6.0, adding Sodium azide, benzoic acid or antibiotic is anti-corrosion
Agent is reagent II, is used for dilute sample and substrate solution；Substrate solution is diluting constant temperature again with reagent II using preceding, and sample is facing measurement
Preceding reagent II dilutes；
(3) setting reaction total volume is required according to amount of liquid in photometer contrastive colours cup when using；First by reagent I constant temperature to not surpassing
37 degree are crossed, then set reaction total volume reagent I is taken to be added in cuvette, 2 ~ 100 ul of product is loaded, is continuously tracked 440 after mixing
Certain absorbing at wavelengths changes between ~ 470 nm；Product formation speed, table are conversed with substrate 4NNPP and its product difference extinction coefficient
Show activity of acid phosphatase.
2. according to described in claim 1, a kind of formula of continuity method measurement activity of acid phosphatase kit, feature also:
(1) sample is such as not required to dilute, substrate solution I is not also diluted, then only needs reagent I without reagent II；
(2) physiological saline also is suitable as reagent II for dilute sample；
(3) in reagent I, the inhibitor for inhibiting specific Acid Phosphatase Isozymes is added, realizes the choosing to specific acid phosphatase
The measurement of selecting property；
(4) in reagent I, in addition to citric acid-sodium citrate buffer solution, other buffers that can cover pH 5.5 are also suitable.
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| CN201811636997.4A CN109576343A (en) | 2018-12-29 | 2018-12-29 | A kind of formula of continuity method measurement activity of acid phosphatase kit |
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| CN201811636997.4A CN109576343A (en) | 2018-12-29 | 2018-12-29 | A kind of formula of continuity method measurement activity of acid phosphatase kit |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5081274A (en) * | 1988-09-19 | 1992-01-14 | Nitto Boseki Co., Ltd. | 4-acyl-2,6-dihalophenyl-phosphoric acid derivatives useful in the determination of acid phosphatase activity |
| CN102706861A (en) * | 2012-05-24 | 2012-10-03 | 宁波美康生物科技股份有限公司 | Acid phosphatase activity determination kit |
| CN102879343A (en) * | 2012-09-21 | 2013-01-16 | 重庆医科大学 | Method for simultaneously measuring activities of various enzymes by using multi-wavelength absorbing single channel |
| CN102977160A (en) * | 2012-11-15 | 2013-03-20 | 重庆医科大学 | Beta-galactosidase chromogenic substrate preparation method using 4-nityl-1-naphthol or derivative of 4-nityl-1-naphthol as chromogenic group and application thereof |
| CN103105391A (en) * | 2012-12-26 | 2013-05-15 | 重庆医科大学 | Novel hydrosulphonyl developing agent and preparation method and application thereof |
| CN103808712A (en) * | 2012-11-05 | 2014-05-21 | 深圳迈瑞生物医疗电子股份有限公司 | Liquid reagent kit applied to acid phosphatase detection and detection method |
-
2018
- 2018-12-29 CN CN201811636997.4A patent/CN109576343A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5081274A (en) * | 1988-09-19 | 1992-01-14 | Nitto Boseki Co., Ltd. | 4-acyl-2,6-dihalophenyl-phosphoric acid derivatives useful in the determination of acid phosphatase activity |
| CN102706861A (en) * | 2012-05-24 | 2012-10-03 | 宁波美康生物科技股份有限公司 | Acid phosphatase activity determination kit |
| CN102879343A (en) * | 2012-09-21 | 2013-01-16 | 重庆医科大学 | Method for simultaneously measuring activities of various enzymes by using multi-wavelength absorbing single channel |
| CN103808712A (en) * | 2012-11-05 | 2014-05-21 | 深圳迈瑞生物医疗电子股份有限公司 | Liquid reagent kit applied to acid phosphatase detection and detection method |
| CN102977160A (en) * | 2012-11-15 | 2013-03-20 | 重庆医科大学 | Beta-galactosidase chromogenic substrate preparation method using 4-nityl-1-naphthol or derivative of 4-nityl-1-naphthol as chromogenic group and application thereof |
| CN103105391A (en) * | 2012-12-26 | 2013-05-15 | 重庆医科大学 | Novel hydrosulphonyl developing agent and preparation method and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| JIZHENGDANG等: "Chromogenic substrate from 4-nitro-1-naphthol for hydrolytic enzyme of neutral or slightly acidic optimum pH: 4-Nitro-1-naphthyl-β-d-galactopyranoside as an example", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| KLAUSLORENTZ: "Continuous monitoring of prostatic acid phosphatase using self-indicating substrates", 《CLINICA CHIMICA ACTA》 * |
| 党济政等: "用4-硝基-1-萘磷酸酯为底物测定碱性磷酸酶活性", 《重庆医科大学学报》 * |
| 冯仁丰等: "《实用医学检验学》", 31 December 1996, 上海：上海科学技术出版社 * |
| 张龙翔等: "《高级生物化学实验选编》", 30 September 1989, 北京：高等教育出版社 * |
| 景一娴等: "低于米氏常数底物浓度下酶动力学参数的测定", 《重庆医科大学学报》 * |
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Effective date of registration: 20210427 Address after: 401332 room 206, building 3, Innovation Productivity Promotion Center, Xiyong micro power park, Shapingba District, Chongqing Applicant after: Chongqing Fulai shark Biotechnology Co.,Ltd. Address before: 401332 2nd Floor, Building 3, Innovative Productivity Promotion Center, Xiyong Science and Technology Third Road, Shapingba District, Chongqing Applicant before: CHONGQING FARSIGHTED BLUE DRAGON (FBD) BIOTECHNOLOGY Co.,Ltd. CHINA |
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Application publication date: 20190405 |