CN106323963A - Multilayer-film dry chemical reagent strip for measuring glutamic-pyruvic transaminase by using enzyme coupled continuous monitoring assay - Google Patents
Multilayer-film dry chemical reagent strip for measuring glutamic-pyruvic transaminase by using enzyme coupled continuous monitoring assay Download PDFInfo
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- CN106323963A CN106323963A CN201610694943.8A CN201610694943A CN106323963A CN 106323963 A CN106323963 A CN 106323963A CN 201610694943 A CN201610694943 A CN 201610694943A CN 106323963 A CN106323963 A CN 106323963A
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- film
- reagent strip
- reaction
- pyruvate transaminase
- concentration
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N2021/3185—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry typically monochromatic or band-limited
Abstract
The invention discloses a multilayer-film dry chemical reagent strip for measuring glutamic-pyruvic transaminase by using enzyme coupled continuous monitoring assay. The reagent strip comprises four layers of film, namely diffusion film, blood filter film (in two layers) and bottom reaction film and is characterized in that corresponding enzyme reactants are adsorbed to the reaction film, pyruvic acid is generated from L-alanine and sodium Alpha-ketoglutarate under the catalysis of glutamic-pyruvic transaminase, the pyruvic acid acts with reduced coenzyme I(NADH) under the catalysis of lactic dehydrogenase (LDH) to generate NAD+, absorbance at 340 nm is decreased, and the amplitude of decrease is associated with the content of the glutamic-pyruvic transaminase. The reagent strip is applicable to the quick measurement of the activity of glutamic-pyruvic transaminase in human whole blood and serum or plasma, is simple and easy to operate, and is easy to clinically apply and popularize.
Description
Technical field
The present invention relates to clinical vitro detection technical field, be specifically related to the assay method of glutamate pyruvate transaminase.
Background technology
Glutamate pyruvate transaminase (ALT), also known as alanine aminotransferase, is catalyzed amino between ALANINE and Pidolidone
Transfer.ALT is widely present in the various tissue of human body, organ, muscle, skeleton, with most in the cytoplasm of liver cell, is liver
Dirty enzyme-specific, and be the most sensitive Testing index of liver function injury by world health organisation recommendations.ALT clinical meaning just exists
In to acute hepatitis B, chronic hepatitis, the HBV person of taking, hepatitis gravis and a series of viral hepatitis such as liver cirrhosis, hepatocarcinoma
Diagnosis and analysis, the rising of ALT only represents that liver may be compromised.
ALT detection method is numerous, and the reagent measuring glutamate pyruvate transaminase clinically can be divided into liquid reagent and drying chemical reagent paper
Two classes.Compared with liquid reagent, chemical reagent bar has conveniently, flexibly, pollute the advantages such as little, easy and simple to handle.
At present, the drying chemical reagent paper of detection glutamate pyruvate transaminase mostly is external product, some complex manufacturing, needs special
Technology；Have there may be some defect, as sample-adding and mensuration are likely to result in interference in the same side.And it is supporting with it
Dry-type biochemical analyser mostly is big-and-middle-sized equipment, and several biochemical projects are concentrated on and measure on an instrument by it.Single measuring
One alanine aminotransferase project time, as the occasions such as mobile blood-collecting carriage, its function and speed can be restricted.Purport of the present invention
There is provided the reagent strip being easy to carry about with one to coordinate independent minicomputer, be more suitable for extensive rapid screening and the family of glutamate pyruvate transaminase
Use.
Summary of the invention
Present disclosure be to provide a kind of easy to use, sensitive can quantitatively determining human whole blood, serum or blood plasma
In the dry chemistry reagent bar of glutamate pyruvate transaminase (ALT).
The dry chemistry reagent bar of the quantitative determination ALT that the present invention provides, for strip, one end is holding area, and the other end is
Test section, its structure such as Fig. 1 and 2, it is made up of upper supporting layer 1, lower supporting layer 2, diffusion barrier 3, hemofiltration film 4,5 and reaction film 6.
Have well 7 in the test section of upper supporting layer 1, the correspondence position of lower supporting layer 2 has instrument connection 8.
In the present invention, reagent strip shell upper supporting layer 1 and lower supporting layer 2 use macromolecule polymeric material to make, and thickness is
0.5mm, upper strata size is 22 × 10mm, and lower floor's size is 45.5 × 10mm.Well and instrument connection on upper lower supporting layer are
Circle, internal diameter is identical, for 4mm.Well 7 is used for dripping sample, and instrument connection 8 is used for observing and measure reaction zone color and extinction
The change of degree.
In the present invention, diffusion barrier 3 is used for accepting sample and spreading downwards, and preferably reaction zone area can accommodate about 10-30 μ
L blood, by two layers of blood separating films, and can be transported to bottom reaction film by test sample product by about 6-20 μ L.
In the present invention, the reaction scheme of employing is enzyme coupling continuous monitoring method, and successive reaction mode is as follows:
In this coupling reaction, the oxidation rate of NADH is proportional with enzymatic activity in specimen, monitors absorbance at 340nm
Rate of descent, thus draw the active unit of ALT.
In the present invention, reaction reagent includes buffer system, TRIS buffer, concentration 250mM, and pH is 7.5；Substrate is L-third
Propylhomoserin and sodium alpha-ketoglutarate, wherein the concentration of ALANINE is 1M-1.5M；The concentration of sodium alpha-ketoglutarate is 50mM-
150mM；The concentration of lactic acid dehydrogenase (LDH) is > 2KU/L；The concentration of NADH > 2mM；The stabilizer of enzyme is bovine serum albumin,
Weight concentration in system is 0.1%-0.25%；Triton X-100 and Hydrazoic acid,sodium salt weight concentration are 0.01%.
For quantitative determination ALT activity, inventor's unit still further developed reflectance spectrophotometer (separate case is applied for a patent), with
Reagent strip of the present invention supports the use.10-30 μ L sample of blood drips on reagent strip, and reaction temperature controls in room temperature, and test divides at 3-5
Complete in clock, instrument automatically report and store experimental result.
The invention has the beneficial effects as follows: the present invention uses the technology for detection glutamate pyruvate transaminase (ALT) of dry type reagent strip, operation
Simple and convenient, be conducive to clinical application and family popularization；The present invention based on the continuous detecting method of enzyme coupling, this inspection
The universal degree of recognition of survey method is high, reacts the sensitiveest；The present invention also added bovine serum albumin, the active group of protection LDH
Group is not destroyed, it is possible to be effectively improved the stability of LDH biochemical reagents.
Accompanying drawing explanation
Fig. 1 is the dry chemistry reagent bar diagram of the present invention.
Fig. 2 is that the dry chemistry reagent bar of the present invention resolves diagram.
Label in figure: 1 is upper supporting layer, 2 is lower supporting layer, and 3 is diffusion barrier, and 4 is rapid glass fibrous filter membrane, and 5 is mixed
Condensating fiber film, 6 is LP reaction film, and 7 is well, and 8 is instrument connection.
Detailed description of the invention
On the lower supporting layer 2 of Fig. 2, the most overlapping diffusion barrier 3, rapid glass fibrous filter membrane 4, mixing fibre
Dimension film 5, LP reaction film 6, then upper supporting layer 1 is pressed on lower supporting layer 2 fixing, obtain the reagent strip such as Fig. 1.
The size of every tunic controls at about 6 × 6mm, after pressing is fixing, it is ensured that well and instrument connection are corresponding in film
The heart, must not have deviation, prevents sample from revealing, affects testing result.
It is as follows that the present invention carries out the detection method of ALT in blood sample:
1. add sample: fluid sample or treated fluid sample are dropped to reagent strip well of the present invention
In；
2. chromatography permeable reactive: stand several minutes and wait that sample chromatography process of osmosis completes and contacts with bottom reaction film,
Carry out enzyme reaction；
3. result interpretation: due to the fact that and reaction film is fixed, the survey its optical signalling changed thereby through instrument
Fixed, interpretation can be realized, thus realize: understand that course of reaction is the most normal and the qualitative and quantitative detection of detected material ALT and dividing
Analysis.
Claims (5)
1. measure the multilayer film dry chemistry reagent bar of glutamate pyruvate transaminase by enzyme coupling continuous monitoring method, including reagent strip shell and interior
Layer test strips, it is characterised in that reagent strip upper surface of outer cover offers well, and bottom offers instrument connection, by upper between holes
Overlap diffusion barrier, hemofiltration film (two-layer) and reaction film the most successively.
Multilayer film dry chemistry reagent bar for measuring glutamate pyruvate transaminase the most according to claim 1, it is characterised in that institute
The diffusion layer stated is the porous diffusion film of one layer of uniform every equal property, and wet-film thickness is 100-300 μm.
Multilayer film dry chemistry reagent bar for measuring glutamate pyruvate transaminase the most according to claim 1, it is characterised in that two
Layers of blood separating film is rapid glass fibrous filter membrane and composite fibre film the most respectively.
Multilayer film dry chemistry reagent bar for measuring glutamate pyruvate transaminase the most according to claim 1, it is characterised in that the end
Layer reaction film is one layer of LP film, and it is adsorbed with reaction reagent；
This reaction reagent includes buffer system, TRIS buffer, concentration 250mM, and pH is 7.5；
Substrate is ALANINE and sodium alpha-ketoglutarate, and wherein the concentration of ALANINE is 1M-1.5M；Sodium alpha-ketoglutarate dense
Degree is 50mM-150mM；
The concentration of lactic acid dehydrogenase (LDH) is > 2KU/L；
The concentration of NADH > 2mM；
The stabilizer of enzyme is bovine serum albumin, and the weight concentration in system is 0.1%-0.25%；
Triton X-100 and Hydrazoic acid,sodium salt weight concentration are 0.01%.
5. according to the multilayer film dry chemistry reagent bar for measuring glutamate pyruvate transaminase described in claim 1 or 4, it is characterised in that
Bottom reaction film there will be the transformation of absorptance with glutamate pyruvate transaminase after reacting, can be recorded by instrument connection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610694943.8A CN106323963A (en) | 2016-08-19 | 2016-08-19 | Multilayer-film dry chemical reagent strip for measuring glutamic-pyruvic transaminase by using enzyme coupled continuous monitoring assay |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610694943.8A CN106323963A (en) | 2016-08-19 | 2016-08-19 | Multilayer-film dry chemical reagent strip for measuring glutamic-pyruvic transaminase by using enzyme coupled continuous monitoring assay |
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| CN106323963A true CN106323963A (en) | 2017-01-11 |
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| CN201610694943.8A Pending CN106323963A (en) | 2016-08-19 | 2016-08-19 | Multilayer-film dry chemical reagent strip for measuring glutamic-pyruvic transaminase by using enzyme coupled continuous monitoring assay |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107727861A (en) * | 2017-08-22 | 2018-02-23 | 厦门依柯利斯医疗科技有限公司 | A kind of pepsin measure kit and assay method |
| CN109580927A (en) * | 2019-01-04 | 2019-04-05 | 杭州联晟生物科技有限公司 | A kind of test card of liver function and preparation method thereof |
| CN109900689A (en) * | 2019-03-28 | 2019-06-18 | 长沙中生众捷生物技术有限公司 | Interference rejection membrane and liver function joint test item |
| CN111197071A (en) * | 2020-01-10 | 2020-05-26 | 杭州联晟生物科技有限公司 | Test card for detecting glutamic-pyruvic transaminase by photochemical method and preparation method thereof |
Citations (4)
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|---|---|---|---|---|
| US5976870A (en) * | 1994-11-09 | 1999-11-02 | Park; Sung-Su | Artificial liver composed of a liver-slice culture apparatus |
| US20020086435A1 (en) * | 2000-12-28 | 2002-07-04 | Fernandez Decastro Aurora L. | Test strip for simultaneous detection of a plurality of analytes |
| CN101597636A (en) * | 2009-07-08 | 2009-12-09 | 重庆医科大学 | Measure the method for target enzymic activity with the maximum instantaneous velocity of conjugate enzyme reaction |
| CN102323399A (en) * | 2011-09-16 | 2012-01-18 | 苏州生物医学工程技术研究所 | Multilayer film dry chemical reagent tablet for detecting alanine aminotransferase |
-
2016
- 2016-08-19 CN CN201610694943.8A patent/CN106323963A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5976870A (en) * | 1994-11-09 | 1999-11-02 | Park; Sung-Su | Artificial liver composed of a liver-slice culture apparatus |
| US20020086435A1 (en) * | 2000-12-28 | 2002-07-04 | Fernandez Decastro Aurora L. | Test strip for simultaneous detection of a plurality of analytes |
| CN101597636A (en) * | 2009-07-08 | 2009-12-09 | 重庆医科大学 | Measure the method for target enzymic activity with the maximum instantaneous velocity of conjugate enzyme reaction |
| CN102323399A (en) * | 2011-09-16 | 2012-01-18 | 苏州生物医学工程技术研究所 | Multilayer film dry chemical reagent tablet for detecting alanine aminotransferase |
Non-Patent Citations (1)
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| 熊振平 等: "《酶工程》", 30 September 1989, 化学工业出版社 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107727861A (en) * | 2017-08-22 | 2018-02-23 | 厦门依柯利斯医疗科技有限公司 | A kind of pepsin measure kit and assay method |
| CN107727861B (en) * | 2017-08-22 | 2019-10-22 | 厦门依柯利斯医疗科技有限公司 | A kind of pepsin assay kit and measuring method |
| CN109580927A (en) * | 2019-01-04 | 2019-04-05 | 杭州联晟生物科技有限公司 | A kind of test card of liver function and preparation method thereof |
| CN109900689A (en) * | 2019-03-28 | 2019-06-18 | 长沙中生众捷生物技术有限公司 | Interference rejection membrane and liver function joint test item |
| CN109900689B (en) * | 2019-03-28 | 2021-12-10 | 复星诊断科技(长沙)有限公司 | Anti-interference membrane and liver function combined test strip |
| CN111197071A (en) * | 2020-01-10 | 2020-05-26 | 杭州联晟生物科技有限公司 | Test card for detecting glutamic-pyruvic transaminase by photochemical method and preparation method thereof |
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Application publication date: 20170111 |