CN110018305A - A kind of aspartate amino transferase Mitochondria Isoenzyme detection kit and application - Google Patents
A kind of aspartate amino transferase Mitochondria Isoenzyme detection kit and application Download PDFInfo
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- CN110018305A CN110018305A CN201910391392.1A CN201910391392A CN110018305A CN 110018305 A CN110018305 A CN 110018305A CN 201910391392 A CN201910391392 A CN 201910391392A CN 110018305 A CN110018305 A CN 110018305A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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Abstract
A kind of aspartate aminotransferase Mitochondria Isoenzyme detection kit, including following component: reagent 1 includes following components: Tris buffer 50mmol/L；ASPARTIC ACID 15mmol/L；Lactic dehydrogenase 0.5mmol/L；Goat-anti people ASTs antibody > 1KU/L；Malic dehydrogenase > 0.8KU/L；Reagent 2 includes following components: α-ketoglutaric acid 130mmol/L；NADH 130mmol/L.
Description
Technical field
The present invention relates to immunodiagnosis fields, and in particular to a kind of aspartate amino transferase Mitochondria Isoenzyme inspection
Test agent box and application method.
Background technique
Aspartate amino transferase (aspartate aminotransferase, AST) is widely present in human body
It is especially the abundantest with liver cell, cardiac muscle cell's content in each histocyte, so AST is usually used in liver diseases and cardiac muscle
The detection of disease.Electrophoretic analysis further divides AST for endochylema type AST (cytosolic AST, c-AST) and line grain
Figure AST (mitochondrial AST, m-AST).The activity of AST is the sum of the activity of c-AST and m-AST in serum.
AST, m-AST, c-AST three are closely related.The detection method of m-AST mainly has immunodepression and enzyme hydrolysis method 2 at present
Kind, but the measurement result of 2 kinds of methods has differences.
This shows the specificity decline of immunodepression m-AST when AST and m-AST is increased, and reason may is that anti-c-
AST antibody is insufficient, and the activity of c-AST does not completely inhibit.Anti- c- has been absolutely proved by additional anti-c-AST antibody test
The problem of AST antibody deficiency, sample after anti-c-AST antibody is additionally added, measured value decline, and with antibody additional amount phase
It closes.Anti- c-AST antibody concentration is one of the major influence factors of m-AST measurement, antibody additional amount and its is stored in reagent
Stability value obtains further investigated in journey.It could also be possible that anti-c-AST antibody specificity is poor, after antibody and c-AST combination not
The activated centre of c-AST is completely covered, causes c-AST that cannot be suppressed completely, m-AST measurement result is higher.
Summary of the invention
It is provided the purpose of the present invention is to solve the above problem.
The invention is realized by the following technical scheme: principle: immunodepression is the anti-c-AST+ c-AST-Ag of monoclonal
→ c-AST-Ag-Ab (c-AST loses activity), then m-AST is detected with performance rate method.L-Aspartic acid+α-ketoglutaric acidm-ASTGrass
Ethyl acetoacetic acid+Pidolidone, oxaloacetic acid+N ADH+ H+ MDHL MALIC ACID+NAD+With NADH 340 nm wavelength absorbance
(A) reduction calculates m-AST vigor.Calculation formula: m-AST (U/L)=Δ A/min × F.
The method applied in the present invention is to have changed the reagent 2 in common m-AST detection kit into comprising two kinds points
Safety pin is to two antigenic determinant c-AST specific monoclonal antibodies, and using it as the specific binding screener of c-AST in serum.
This way can be played the role of with lesser concentration close c-AST it is active, it is more efficient than usually used polyclonal antibody,
Polyclonal antibody for the Multiple Antibodies of multiple antigenic determinants due to mixing, and specificity is not high when closing, to antibody
Concentration requirement is very high, if the polyclonal antibody for encountering the batch is not high enough for the concentration of specific position, will cause closing
Not enough, cause error when measurement, and can be assessed according to actual enzyme concentration range using monoclonal antibody, according to maximum
Demand carries out the active closing of c-AST, and is all specificity efficiently for enzyme active sites, and efficiency further increases.
Use the specific monoclonal antibody for two antigenic determinants of c-AST albumen as the screener of the protein active,
Be used to prepare m-AST detection kit, used two antigenic determinants be respectively be directed to two of c-AST protein sequence it is anti-
Former determinant is two segments of NHEYLPILGLAEFRSCASRLAL and KQIASVMKHRFLFPFFDSAYQG respectively, this two
The monoclonal antibody that a segment prepares, specificity is very high, can obtain height when in use with smaller concentration
The effect of effect shielding c-AST enzymatic activity can play m- in individually detection serum in measuring serum when m-AST activity
The active effect of AST.
Beneficial effect
The usage amount that antibody can be controlled plays the role of save the cost and improves the measuring value accuracy of kit.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail.Following embodiment be only it is exemplary, only
To do further detailed description to technical solution of the present invention, those skilled in the art should understand that, not
In the spirit and scope for deviateing technical solution of the present invention, modifying or replaceing for technical solution progress should all be covered in the present invention
Claims in.
The detection kit prepared by the present invention of embodiment 1.
Reagent 1 includes following components: Tris buffer 50mmol/L；ASPARTIC ACID 15mmol/L；Lactic dehydrogenase
0.5mmol/L；1 > 1KU/L of goat-anti people c-AST antibody；2 > 1KU/L of goat-anti people c-AST antibody；Malic dehydrogenase > 0.8KU
/L；Reagent 2 includes following components: α-ketoglutaric acid 130mmol/L；NADH 130mmol/L.Wherein goat-anti people c-AST antibody 1
Combination antigenic determinant be NHEYLPILGLAEFRSCASRLAL；The combination antigenic determinant of goat-anti people c-AST antibody 2 is
KQIASVMKHRFLFPFFDSAYQG；
The kit that embodiment 2. is prepared using polyclonal antibody
Reagent 1 includes following components: Tris buffer 50mmol/L；ASPARTIC ACID 15mmol/L；Lactic dehydrogenase
0.5mmol/L；Polyclonal goat-anti people m-AST antibody > 1KU/L；Malic dehydrogenase > 0.8KU/L；Reagent 2 includes with the following group
Point: α-ketoglutaric acid 130mmol/L；NADH 130mmol/L.
The monoclonal antibody that embodiment 3. uses other sites to prepare is as screener.
Reagent 1 includes following components: Tris buffer 50mmol/L；ASPARTIC ACID 15mmol/L；Lactic dehydrogenase
0.5mmol/L；1 > 1KU/L of goat-anti people c-AST antibody；2 > 1KU/L of goat-anti people c-AST antibody；Malic dehydrogenase > 0.8KU
/L；Reagent 2 includes following components: α-ketoglutaric acid 130mmol/L；NADH 130mmol/L.Wherein goat-anti people c-AST antibody 1
Combination antigenic determinant be LRARLEA；The combination antigenic determinant of goat-anti people c-AST antibody 2 is WAIRYFVSE；
The kit that embodiment 4. is prepared using the stabilizer of monoclonal antibody.
Reagent 1 includes following components: Tris buffer 50mmol/L；ASPARTIC ACID 15mmol/L；Lactic dehydrogenase
0.5mmol/L；Goat-anti people ASTs antibody > 1KU/L；Malic dehydrogenase > 0.8KU/L；Reagent 2 includes following components: α -one
Glutaric acid 130mmol/L；NADH 130mmol/L；The concentration in reagent for being added to stabilizer mannose is 0.1g/L, sweet
Oil concentration in reagent is 0.05g/L.
1. betweenrun precision of experimental example: taking a kind of concentration is 15U/L serum specimen, by withinrun precision requirement to above-mentioned
The kit of 4 embodiments preparation is tested, and criticizes interior CV result such as following table as the result is shown.
| Embodiment | 1 | 2 | 3 | 4 |
| CV value | 3.06% | 5.78% | 6.69% | 3.06% |
| CV value (after 30 days) | 4.11% | 7.23% | 8.41% | 3.12% |
2. sensitivity for analysis of experimental example: according to testing result when measured object concentration is 20 U/L, 4 kinds of kits prepared
Absorbance change value is all between 0.008-0.050.Meet national standard.
Sequence table
<110>biotech inc Wuhan Sheng Zhiyuan
<120>a kind of aspartate amino transferase Mitochondria Isoenzyme detection kit and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 2
<211> 22
<212> PRT
<213>homo sapiens (Homo sapiens)
<400> 2
Asn His Glu Tyr Leu Pro Ile Leu Gly Leu Ala Glu Phe Arg Ser Cys
1 5 10 15
Ala Ser Arg Leu Ala Leu
20
<210> 3
<211> 22
<212> PRT
<213>homo sapiens (Homo sapiens)
<400> 3
Lys Gln Ile Ala Ser Val Met Lys His Arg Phe Leu Phe Pro Phe Phe
1 5 10 15
Asp Ser Ala Tyr Gln Gly
20
Claims (6)
1. a kind of aspartate aminotransferase Mitochondria Isoenzyme detection kit, including reagent 1, reagent 2, m-AST albumen
Standard items, it is characterised in that: reagent 2 includes two kinds respectively for two antigenic determinant c-AST specific monoclonal antibodies as serum
The specific binding screener of middle c-AST.
2. 2 kinds as described in claim 1 of c-AST specific monoclonal antibody, which is characterized in that the epitope sequence combined
For shown in SEQ ID1 and SEQ ID2.
3. aspartate aminotransferase Mitochondria Isoenzyme kit as claimed in claim 2, it is characterised in that: reagent 1
Including following components: Tris buffer 50mmol/L；ASPARTIC ACID 15mmol/L；Lactic dehydrogenase 0.5mmol/L；Goat-anti
People c-ASTs antibody > 1KU/L；Malic dehydrogenase > 0.8KU/L.
4. aspartate aminotransferase Mitochondria Isoenzyme kit as claimed in claim 2, it is characterised in that: reagent 2
Including following components: α-ketoglutaric acid 130mmol/L；NADH 130mmol/L.
5. kit as shown in claim 1, it is characterised in that: reagent 1 and reagent 2 all also contain antibody stabilization agent mannose
And glycerol.
6. antibody stabilization agent as claimed in claim 5: the concentration in reagent of mannose is 0.1g/L, and glycerol is in reagent
Concentration is 0.05g/L.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910391392.1A CN110018305B (en) | 2019-05-12 | 2019-05-12 | Aspartate aminotransferase mitochondrial isozyme detection kit and application |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910391392.1A CN110018305B (en) | 2019-05-12 | 2019-05-12 | Aspartate aminotransferase mitochondrial isozyme detection kit and application |
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| CN110018305A true CN110018305A (en) | 2019-07-16 |
| CN110018305B CN110018305B (en) | 2020-12-08 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113164110A (en) * | 2019-09-17 | 2021-07-23 | 诺尔生物医药有限公司 | System and method for measuring liver enzyme levels in blood |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4732857A (en) * | 1984-01-12 | 1988-03-22 | Sawao Murao | Process for producing enzyme capable of inactivating cytosolic aspartate aminotransferase isozyme |
| 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 |
| CN102010890A (en) * | 2009-09-08 | 2011-04-13 | 北京迪迈医学诊断技术有限公司 | Kit for determining mitochondria aspartate aminotransferase |
| CN104459158A (en) * | 2014-12-22 | 2015-03-25 | 宁波美康生物科技股份有限公司 | Aspartate transaminase mitochondrial isozyme detection kit |
| 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 |
-
2019
- 2019-05-12 CN CN201910391392.1A patent/CN110018305B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4732857A (en) * | 1984-01-12 | 1988-03-22 | Sawao Murao | Process for producing enzyme capable of inactivating cytosolic aspartate aminotransferase isozyme |
| 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 |
| CN102010890A (en) * | 2009-09-08 | 2011-04-13 | 北京迪迈医学诊断技术有限公司 | Kit for determining mitochondria aspartate aminotransferase |
| CN104459158A (en) * | 2014-12-22 | 2015-03-25 | 宁波美康生物科技股份有限公司 | Aspartate transaminase mitochondrial isozyme detection kit |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 保方 等: "免疫抑制法检测m-AST及其应用", 《现代检验医学杂志》 * |
| 荣墨克 等: "ELlSA法测定线粒体天门冬氨酸氨基转移酶", 《吉林大学学报（医学报）》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113164110A (en) * | 2019-09-17 | 2021-07-23 | 诺尔生物医药有限公司 | System and method for measuring liver enzyme levels in blood |
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Inventor after: Hua Quangao Inventor after: Gong Ting Inventor after: Wu Nianfen Inventor after: Wu Weijiao Inventor after: Huang Ai Inventor after: Liang Yan Inventor after: Shen Hexiao Inventor after: Zhao Chang Inventor after: Xu Chunlei Inventor before: Hua Quangao Inventor before: Shen Hexiao Inventor before: Xu Chunlei |
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