CN104459158A - Aspartate transaminase mitochondrial isozyme detection kit - Google Patents
Aspartate transaminase mitochondrial isozyme detection kit Download PDFInfo
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- CN104459158A CN104459158A CN201410805113.9A CN201410805113A CN104459158A CN 104459158 A CN104459158 A CN 104459158A CN 201410805113 A CN201410805113 A CN 201410805113A CN 104459158 A CN104459158 A CN 104459158A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention discloses an aspartate transaminase mitochondrial isozyme detection kit, comprising: reagents 1: 0.02-0.5mol/L of buffer solution of which pH value is 6.5-8.0, 0.15-0.3mol/L of L-potassium aspartate, 0.3-5KU/L of malic dehydrogenase, 0.3-5KU/L of lactic dehydrogenase, 5-20mL/L of human cAST antibody, 0.01-3% of antibody stabilizer, 0.01-10% of enzyme stabilizer and 0.01-1% of preservative; and reagents 2: 0.02-0.5mol/L of buffer solution of which pH value is 7.0-9.0, 0.005-0.02mol/L of alpha-ketoglutarate, 0.1-0.5mmol/L of reduced coenzyme I, 0.01-1% of preservative and 0.01-10% of reduced coenzyme I stabilizer. The kit has the advantage of good stability.
Description
Technical field
The present invention relates to technical field of medical examination, be specifically related to a kind of aspartate aminotransferase Mitochondria Isoenzyme (m-AST or mAST Mitochondrial form aspartate amino transferase) detection kit.
Background technology
Aspartate aminotransferase (Aspartate aminotransferase, AST) is extensively present in the various histocyte of human body, especially abundant with liver, heart tissue's cell content, and in normal human serum, AST activity is very low.In human body there are two kinds of isodynamic enzymes in AST, and one is cAST, is mainly derived from cytoplasm, and another kind is mAST, is present in mitochondria.These two kinds of isodynamic enzymes are variant in genomic constitution, all different in amino acid composition, immunological characteristic and half life period in vivo etc.
When the slight pathology of body, histiocytic permeability of cell membrane increases, and cAST permeate through cell membranes enters in hematological system, but mAST has the protection of two-layer mitochondrial membrane, is not easily released into blood; And when body serious disease, there is meronecrosis, mitochondria disintegration, the mAST be attached on mitochondria can discharge into rapidly in blood.Therefore, serum mAST activity can reflect histiocytic degree of necrosis, if detect high-level mAST in serum, then shows cardiac muscle, liver is badly damaged; The half life period of mAST in blood is shorter than cAST in addition, and when cell no longer destroys or repairs, in serum, mAST is down to normal level very soon, and therefore mAST is again the evaluation index of cardiac muscle, liver damage prognosis, has important value to the diagnosis of disease, treatment.
At present, in serum, mAST measures and mainly contains electrophoresis, chromatography and immunodepression etc., especially possesses advantage that is special, accurate, fast and convenient, that can be used for Aulomatizeted Detect with immunodepression, is suitable for the application of clinical extensive sample.Its Cleaning Principle is: in people source cAST antibody and serum sample, cAST effect forms immune complex, make cAST activity inhibited, and mAST activity is unaffected, mAST can with the reaction coupling of malic dehydrogenase (MDH) catalysis, make reduced coenzyme Ⅰ (NADH) be oxidized to NAD
+, by detecting the speed that certain wave strong point NADH absorbance declines, can be calculated the activity of mAST.Reaction equation is as follows:
From above-mentioned reaction principle, people source cAST antibody is the critical materials of mAST determination of activity system, and can people source cAST antibody becomes the key of mAST activity in Accurate Measurement human serum sample.But, according to this area general knowledge, when antibody is with solution state long-term storage, the phenomenons such as the generation of chemolysis thing, the generation of insoluble agglutinator, the generation of solubility associated matter can be there is, thus cause the biologically active of antibody greatly to reduce, therefore antibody is most to save as the best under-20 DEG C of environment, in order to keep the activity of antibody, can only provisional configuration kit in use, and this is not obviously suitable for commercial liquid reagent box.
Summary of the invention
The present invention is directed to the above-mentioned deficiency of prior art, a kind of aspartate aminotransferase Mitochondria Isoenzyme detection kit of excellent in stability is provided.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of aspartate aminotransferase Mitochondria Isoenzyme detection kit, and this kit is made up of following two kinds of liquid reagents, wherein:
Reagent 1:
Reagent 2:
Wherein, damping fluid in described reagent 1 or 2 can be one or more in TRIS buffer, two (2-hydroxyethanesulfonic acid) damping fluid of piperazine-N, N-, N-2-hydroxyethyl piperazine-N '-ethanesulfonic acid buffer, N-tri-(methylol) methylamino-2-hydroxy-propanesulfonic acid damping fluid, 4-(2-hydroxyethyl)-1-piperazine propane sulfonic acid damping fluid, 3-morpholine-2-hydroxypropionate sodium damping fluid;
Described antibody stabilization agent can be the one during glycocoll and citric acid, glycocoll and heparin, citric acid and histidine, heparin and histidine combine, the combination of preferred histidine and citric acid, the weight proportion between said sequence combination is: histidine: citric acid=10 ~ 100:1;
Described enzyme stabilizers is one or several in trehalose, sucrose, bovine serum albumin(BSA), glycerine, Tween 80;
Described antiseptic is NaN
3or the one in proclin300;
Described reduced coenzyme Ⅰ stabilizing agent is one or more in trehalose, bovine serum albumin(BSA), glycerine, Tween 80.
Advantage of the present invention and beneficial effect: mAST detection kit of the present invention is by adding antibody stabilization agent, to improve in kit people source cAST antibody in the stability of liquid condition, enzyme stabilizers and reduced coenzyme Ⅰ stabilizing agent is added additionally by combining, therefore more effectively ensure that the stability of kit entirety, greatly meet the demand of clinical detection and diagnosis.
Specific embodiment
Below in conjunction with embodiment, the present invention is further described in detail, but is not limited to this.
Embodiment 1
A kind of mAST detection kit, comprises reagent 1 and reagent 2, wherein:
Reagent 1:
Reagent 2:
Embodiment 2
A kind of mAST detection kit, comprises reagent 1 and reagent 2, wherein:
Reagent 1:
Reagent 2:
Below the stability of the embodiment of the present invention 1 gained reagent is described.
1, Antibody stability:
Be configured to down three group reagents by the following method:
Organize 1.: according to recipe configuration reagent described in embodiment 1, and in 2 ~ 8 DEG C of storages 18 months;
Organize 2.: except not adding antibody stabilization agent, all the other are according to recipe configuration reagent described in embodiment 1, and in 2 ~ 8 DEG C of storages 18 months;
Organize 3.: after 18 months, newly configure reagent (antibody added is-20 DEG C of frozen same a collection of antibody) according to filling a prescription described in embodiment 1.
Above-mentioned three group reagents are placed on automatic clinical chemistry analyzer, location parameter is set in accordance with the following methods and the activity of Simultaneously test 10 parts of human serum mAST: temperature of reaction 37 DEG C, test predominant wavelength 340nm, test commplementary wave length 405nm, tested serum sample/blank with reagents ratio is: sample/contrast: reagent 1: reagent 2=18uL:240uL:60uL, reacts for falling reaction.Method of testing is: sample/contrast first mixes with reagent 1 hatches 5min, add reagent 2 afterwards, reading is started after in 1.5min, and under mensuration wavelength, monitor the change of each sample absorbance in 3min continuously, calculate the absorbance changing value △ A/min of average minute clock, and according to following formulae discovery mAST activity value: mAST activity (U/L)=(△ A
measure/ min-△ A
blank/ min) × F (2840.3).Measurement result is as shown in table 1.
Table 1mAST detection kit Antibody stability result
As can be seen from table 1 result: the reagent not adding antibody stabilization agent is in storage after 18 months, measured result exceeds about 10% than the reagent of new configuration, the antibody illustrating in liquid reagent is deposited 18 months artifact activity and is decreased, and rejection ability declines to some extent; And with the addition of the reagent of antibody stabilization agent, the result of mensuration with newly join reagent and still can be consistent (± 3%), show that antibody stabilization agent can protect the biologically active of antibody in liquid reagent effectively, improve the stability of antibody.
2, thermal stability:
According to recipe configuration reagent described in embodiment 1, be placed in heat damage 7d under 37 DEG C of water bath, measure and record the blank absorbency changing value of reagent in 7d, and measuring the mAST value of 10 parts of human serum samples in 7d.
Table 2mAST detects each Blank absorbance values in reagent 37 DEG C of water-bath 7d
Determination of serum result after table 3mAST detection reagent 37 DEG C of water-bath 7d
As can be seen from table 2 and table 3 result, 7d placed by reagent 37 DEG C, and blank absorbency is without significant change, and determination of serum result is close to contrast, and result is good.
People source of the present invention cAST antibody is commercially available prod, as Thermo scientific company the GOT1Antibodies that sold.
The above embodiment of the present invention can not be used for limiting the present invention to explanation of the present invention, and any change in the implication suitable with claims of the present invention and scope, all should think to be included in the scope of claims.
Claims (7)
1. an aspartate aminotransferase Mitochondria Isoenzyme detection kit, is characterized in that: this kit is made up of reagent 1 and reagent 2 two kinds of liquid reagents, wherein:
Reagent 1:
Reagent 2:
2. aspartate aminotransferase Mitochondria Isoenzyme detection kit according to claim 1, it is characterized in that: the damping fluid in described reagent 1 or 2 is one or more in TRIS buffer, two (2-hydroxyethanesulfonic acid) damping fluid of piperazine-N, N-, N-2-hydroxyethyl piperazine-N '-ethanesulfonic acid buffer, N-tri-(methylol) methylamino-2-hydroxy-propanesulfonic acid damping fluid, 4-(2-hydroxyethyl)-1-piperazine propane sulfonic acid damping fluid, 3-morpholine-2-hydroxypropionate sodium damping fluid.
3. aspartate aminotransferase Mitochondria Isoenzyme detection kit according to claim 1, is characterized in that: described antibody stabilization agent is the one during glycocoll and citric acid, glycocoll and heparin, citric acid and histidine, heparin and histidine combine.
4. aspartate aminotransferase Mitochondria Isoenzyme detection kit according to claim 3, is characterized in that: described antibody stabilization agent is the combination of histidine and citric acid.
5. aspartate aminotransferase Mitochondria Isoenzyme detection kit according to claim 1, is characterized in that: described enzyme stabilizers is one or several in trehalose, sucrose, bovine serum albumin(BSA), glycerine, Tween 80.
6. aspartate aminotransferase Mitochondria Isoenzyme detection kit according to claim 1, is characterized in that: described antiseptic is NaN
3or the one in proclin300.
7. aspartate aminotransferase Mitochondria Isoenzyme detection kit according to claim 1, is characterized in that: described reduced coenzyme Ⅰ stabilizing agent is one or more in trehalose, bovine serum albumin(BSA), glycerine, Tween 80.
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| CN201410805113.9A CN104459158B (en) | 2014-12-22 | 2014-12-22 | Aspartate aminotransferase Mitochondria Isoenzyme detection kit |
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| CN201410805113.9A CN104459158B (en) | 2014-12-22 | 2014-12-22 | Aspartate aminotransferase Mitochondria Isoenzyme detection kit |
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| CN104459158B CN104459158B (en) | 2016-06-22 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104865214A (en) * | 2015-05-08 | 2015-08-26 | 浙江蓝森生物科技有限公司 | Method, reagent and kit for quantitative determination of mitochondrial AST activity in human serum |
| CN106244671A (en) * | 2016-08-30 | 2016-12-21 | 山东博科生物产业有限公司 | A kind of stable aspartate aminotransferase checkout and diagnosis detection kit |
| CN107860729A (en) * | 2017-11-08 | 2018-03-30 | 北京北检·新创源生物技术有限公司 | A kind of stable reagent for being used to determine glutamic-oxalacetic transaminease |
| CN110018305A (en) * | 2019-05-12 | 2019-07-16 | 武汉生之源生物科技股份有限公司 | A kind of aspartate amino transferase Mitochondria Isoenzyme detection kit and application |
| CN110079581A (en) * | 2019-05-14 | 2019-08-02 | 天津中成佳益生物科技有限公司 | A kind of detection kit and detection method of aspartate amino transferase Mitochondria Isoenzyme |
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| JPS60169766A (en) * | 1984-02-14 | 1985-09-03 | Sugiura Shinyaku Kaihatsu Kenkyusho:Kk | Reagent for fractional measurement of soluble fraction aspartic acid aminotransferase and mitochondria aspartic acid aminotransferase |
| US4769323A (en) * | 1984-01-12 | 1988-09-06 | Sawao Murao | Method for fractional determination of aspartate aminotransferase isozymes, and composition therefor |
| CN1546683A (en) * | 2003-12-15 | 2004-11-17 | 上海北加生化试剂有限公司 | Aspartic transaminase mitochondrion isozyme m type measurement method |
| CN102399851A (en) * | 2011-10-25 | 2012-04-04 | 宁波美康生物科技有限公司 | Sialidase detection kit |
| CN102946858A (en) * | 2010-05-10 | 2013-02-27 | 英塔斯生物制药有限公司 | Liquid formulation of polypeptides containing an Fc domain of an immunoglobulin |
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2014
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Patent Citations (5)
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| US4769323A (en) * | 1984-01-12 | 1988-09-06 | Sawao Murao | Method for fractional determination of aspartate aminotransferase isozymes, and composition therefor |
| JPS60169766A (en) * | 1984-02-14 | 1985-09-03 | Sugiura Shinyaku Kaihatsu Kenkyusho:Kk | Reagent for fractional measurement of soluble fraction aspartic acid aminotransferase and mitochondria aspartic acid aminotransferase |
| CN1546683A (en) * | 2003-12-15 | 2004-11-17 | 上海北加生化试剂有限公司 | Aspartic transaminase mitochondrion isozyme m type measurement method |
| CN102946858A (en) * | 2010-05-10 | 2013-02-27 | 英塔斯生物制药有限公司 | Liquid formulation of polypeptides containing an Fc domain of an immunoglobulin |
| CN102399851A (en) * | 2011-10-25 | 2012-04-04 | 宁波美康生物科技有限公司 | Sialidase detection kit |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104865214A (en) * | 2015-05-08 | 2015-08-26 | 浙江蓝森生物科技有限公司 | Method, reagent and kit for quantitative determination of mitochondrial AST activity in human serum |
| CN106244671A (en) * | 2016-08-30 | 2016-12-21 | 山东博科生物产业有限公司 | A kind of stable aspartate aminotransferase checkout and diagnosis detection kit |
| CN107860729A (en) * | 2017-11-08 | 2018-03-30 | 北京北检·新创源生物技术有限公司 | A kind of stable reagent for being used to determine glutamic-oxalacetic transaminease |
| CN107860729B (en) * | 2017-11-08 | 2022-03-11 | 北京北检·新创源生物技术有限公司 | Stable reagent for determining glutamic-oxalacetic transaminase |
| CN110018305A (en) * | 2019-05-12 | 2019-07-16 | 武汉生之源生物科技股份有限公司 | A kind of aspartate amino transferase Mitochondria Isoenzyme detection kit and application |
| CN110018305B (en) * | 2019-05-12 | 2020-12-08 | 武汉生之源生物科技股份有限公司 | Aspartate aminotransferase mitochondrial isozyme detection kit and application |
| CN110079581A (en) * | 2019-05-14 | 2019-08-02 | 天津中成佳益生物科技有限公司 | A kind of detection kit and detection method of aspartate amino transferase Mitochondria Isoenzyme |
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| CN104459158B (en) | 2016-06-22 |
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Address after: 315104 Ningbo, Yinzhou, Central District, No. Qiming South Road, No. 299 Patentee after: Meikang biological Polytron Technologies Inc Address before: 315104 Ningbo, Yinzhou, Central District, No. Qiming South Road, No. 299 Patentee before: Ningbo Meikang Biotechnology Co., Ltd. |
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Inventor after: Zou Bingde Inventor after: Zou Jihua Inventor after: Lu Huixian Inventor after: Huang Xinglei Inventor before: Zou Bingde Inventor before: Zou Jihua Inventor before: Lu Huixian Inventor before: Huang Xinlei |
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