CN102586397A - Enzymatic detection adenosine deaminase kit - Google Patents
Enzymatic detection adenosine deaminase kit Download PDFInfo
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- CN102586397A CN102586397A CN2011100042871A CN201110004287A CN102586397A CN 102586397 A CN102586397 A CN 102586397A CN 2011100042871 A CN2011100042871 A CN 2011100042871A CN 201110004287 A CN201110004287 A CN 201110004287A CN 102586397 A CN102586397 A CN 102586397A
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- reagent
- adenosine deaminase
- enzyme
- kit
- test kit
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Abstract
The invention discloses an enzymatic detection adenosine deaminase kit which comprises a reagent 1 and a reagent 2. Enzymatic compound accelerant is added into the enzymatic detection adenosine deaminase kit, so that the sensitivity on a reaction is improved, and the reaction of adenosine deaminase can be accelerated; a composite inhibitor is selected for use, so that the specificity of the reaction is guaranteed; and enzyme protecting agent is added into the kit, so that the enzyme in the reagent can be better protected, and the stability of the reagent can be improved. The enzymatic detection adenosine deaminase kit has more obvious protection function on the enzyme; and compared with a reagent which is not added with the protecting agent, the survival rate of residual enzyme is increased by 51% after 12 months. Therefore, the enzymatic detection adenosine deaminase kit has better clinical application prospect.
Description
Technical field:
The present invention relates to biological reagent, be specifically related to a kind of detection kit, relate in particular to a kind of enzyme process and detect the adenosine deaminase test kit.
Background technology:
Adenosine deaminase (ADA) is an important enzyme important in the purine nucleoside metabolism, belongs to the sulfydryl enzyme, and each molecule contains 2 sulfhydryl-group activities at least, thereby it can suppress chlorine mercury formic acid fully.In addition, ADA can change inosine into by the catalysis adenosine, generates xanthoglobulin through the nucleoside phosphorylase effect again, and its metabolic end product is a uric acid.ADA is distributed widely in each tissue of human body, and the highest with content in thymus gland, spleen and other Lymphoid tissues, it is lower that liver, lung, kidney and skeletal muscle etc. are located content.ADA mainly is present in red corpuscle, granulocyte and lymphocyte in the blood, and its activity is about 40～70 times of serum, and this enzyme is higher than bone-marrow-derived lymphocyte activity at the T lymphocyte.At present, adenosine deaminase (ADA) mainly contains in Clinical Application:
One, is used for the diagnosis of liver and gall diseases
1. acute hepatitis patient ADA and ALT all significantly raise.The rising of hepatitis B patient ALT is greater than ADA, but not the rising of first non-hepatitis B patient ADA is greater than ALT, and the active extremely rising person of Fei Jiafeiyiganyan patient ADA is prone to change into chronic persistent hepatitis more.2. chronic hepatitis patient ADA activity all raises, and chronic active hepatitis raises more very than non-active type.3. active significantly rising of hepatic fibrosis, liver cirrhosis and chronic hepatitis patient ADA and ALT does not raise is so measure ALT and ADA has more clinical value simultaneously.4. obstructive jaundice ADA does not raise, and raises and hepatocellular jaundice ADA is active, therefore measures the differential diagnosis that serum ADA helps the jaundice type.
Two, be used for the diagnosis of tuberculous pleuritis and tuberculous peritonitis: pleural effusions and ascites ADA determination of activity, can distinguish the character of pleural effusions and ascites.The ADA activity is active apparently higher than the ADA in cancer and the heart failure property hydrothorax in tuberculous pleuritis and the tuberculous peritonitis patient pleural effusions and ascites; And the ratio of ADA/ serum ADA is greater than 1, so measure the reliable and effective index that ascites pleural fluid and serum ADA activity and ratio are diagnosis tuberculous pleuritis and tuberculous peritonitis in the hydrops.
Three, typhoid: typhoid fever patient ADA is active significantly to raise; Apparently higher than the fever patient of non-typhoid fever, ADA susceptibility reaches 100%, specificity 92.3%; The typhoid fever their early stage can be higher than 2～4 times of normal peoples; Crisis and catabasis still continue to be in high level, to reducing gradually in the 4th week of the course of disease, so the ADA detection is very valuable to the early diagnosis of typhoid.
Four, other disease: like infectious monocytosis, miliary tuberculosis, rheumatic fever, hemolytic anemia, white blood disease and part tumour patient, the rising that serum ADA can be in various degree.Combined immunodeficiency disease (SCID), each histocyte ADA activity of whole body all lacks.ADA is active in tuberculous meningitis patient's the cerebrospinal fluid raises, and other meningitis does not raise, so the ADA determination of activity can be differentiated tuberculous meningitis in the cerebrospinal fluid.
The active detection method of ADA generally has biochemical process and active nucleus method, and the latter is highly sensitive, but radiocontamination is arranged, so the biochemical method application is more extensive.
Colourimetry: adopt coupling reaction to measure, calculate that with the ammonia amount that discharges in the unit time ADA is active.These methods of the many employings of China at present, its advantage is for need not specific apparatus and reagent; But be only applicable to manual operations, and long reaction time, interfering factors is also more, is difficult to obtain the rapid and precise result.
The enzyme coupling method coupling, generally use purine nucleoside phosphorylase (Purine nucleosidehosphorylase, PNP) and XOD (Xanthine Oxidase, XOD) px (PEROXIDASE, PEO).Hydrogen peroxide generates the quinone dyestuff with N acetaldehyde one N, one 3 mono methyl aniline 99.5004323A8uritys and aminoantipyrene reaction under the px effect, kinetics continuous monitoring quinone concentration is measured the ADA activity.This method specificity is good, and precision is high, good reproducibility, and linearity range can reach 200U/I, significantly is superior to known other chemical methods.Reagent stability is better, stores under the 2 ℃～8C temperature and can stablize 1 year, and this method is used more.
The enzyme process principle:
Adenosine
ADA＞Trophicardyl+NH
4 +
Trophicardyl+Pi
PNP＞xanthoglobulin+ribose-1-phosphoric acid
Xanthoglobulin
XOD＞uric acid+H
2O
2
H
2O
2+ 4-AAP+EHSPT
POD＞red quinone derivative+H
2O
But some shortcoming of this detection method at present, PNP (purine nucleoside phosphorylase) reagent cost an arm and a leg, use price high, and PNP reagent is unstable, and reagent react speed is slow, and reagent receives the interference of SEAP etc. easily.
Summary of the invention:
Technical problem to be solved by this invention is to overcome above-mentioned weak point, and a kind of acceleration adenosine deaminase reaction is provided, and the enzyme process that improves reagent stability detects the reagent of adenosine deaminase.
The invention provides a kind of enzyme process and detect the adenosine deaminase test kit, this test kit is made up of following reagent 1 and reagent 2:
Reagent 1: (3L)
Damping fluid 10-200mmol/L
Toos (N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-monomethylaniline sodium salt)
1-100mmol/L
Triton x-100 (Triton 100) 0.01-20ml/L
Sodium Benzoate 20-100mmol/L
FeCl3 1-100mmol/L
PNP (purine nucleoside phosphorylase) 10-20u/L
Xot (XOD) 20-30u/L
Peo (px) 200-300u/L
Glycerose 10-100g/L
Sodium arseniate 10-100g/L
Reagent 2: (1L)
Sodium phosphate, dibasic 10-200mmol/L
KCl 10-500mmol/L
N.F,USP MANNITOL 10-200mmol/L
Proclin300 (biological preservative) 0.01-0.5ml/L
Adenosine 10-200mmol/L
4-is amino to be replaced than woods 1-5mmol/L.
Said reagent 1 damping fluid is selected from TRIS (Tutofusin tris tris (hydroxymethyl) aminomethane), MOPS (3-(N-morpholine) propanesulfonic acid 3-(N-morpholino) propanesulfonic acid) or HEPES (4-HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).
Enzyme process of the present invention detects the adenosine deaminase test kit, and in use, reagent 1 adds reagent 2 after adding the detection sample again, can detect.
Another object of the present invention has provided the preparation method of enzyme process detection adenosine deaminase test kit: comprise the following steps:
(1) preparation reagent 1: (3L)
(1) elder generation is incorporated as the deionized water of total amount 80% in container;
(2) add damping fluid, toos, Triton x-100 (Triton 100) successively
Sodium Benzoate, FeCl3, Glycerose, sodium arseniate;
(3) add enzyme: PNP (purine nucleoside phosphorylase),
Xot (XOD), Peo (px);
(4) adding remaining deionized water at last mixes;
(2) preparation reagent 2: (1L)
In container, add successively Sodium phosphate, dibasic, KCl, N.F,USP MANNITOL,
Proclin300, adenosine, amino the replacing of 4-mix than woods.
The present invention is based on following principle:
(1) select for use promotor to quicken the reaction of adenosine deaminase, wherein the compound accelerant of enzyme is FeCl3, Sodium Benzoate, TrionX-100 (tensio-active agent);
(2) select composite inhibitor for use, guarantee the specificity of reacting, suppress the interference of SEAP, composite inhibitor is: Glycerose;
(3) add special enzymatic protective reagent such as N.F,USP MANNITOL, guaranteed the stability of reagent.
Test kit of the present invention detects effective:
(1) 0.1M FeCl
3, Sodium Benzoate, Triton x-100 improve as the sensitivity of compounding activation agent to reaction, the time shortens.
(2) test kit of the present invention has very strong provide protection to the enzyme in the reagent, and is As time goes on more obvious to the provide protection meeting of enzyme, adds protectant reagent after 12 months and compares with not adding protectant reagent, and remaining enzyme motility rate raises 57%.
Embodiment:
Embodiment 1 preparation enzyme process detects the adenosine deaminase test kit
Form:
Reagent 1: (3L)
TRIS damping fluid 150mmol
toos 6mmol
Triton?x-100 6ml
Sodium Benzoate 60mmol
FeCl3 9mmol
PNP 30u
Xot 90u
Peo 660u
Glycerose 60g
Sodium arseniate 240g
Reagent 2: (1L)
Sodium phosphate, dibasic 50mmol
KCl 10mmol
N.F,USP MANNITOL 20mmol
proclin300 0.5ml
Adenosine 15mmol
4-is amino to be replaced than woods 3mmol
Preparation:
(1) preparation reagent 1: (3L)
(1) elder generation adds the deionized water of 2.4L in container;
(2) add TRIS damping fluid, toos, Triton x-100 (Triton 100) successively
Sodium Benzoate, FeCl3, Glycerose, sodium arseniate;
(3) add enzyme: PNP (purine nucleoside phosphorylase),
Xot (XOD), Peo (px);
(4) adding remainder water at last mixes;
(2) preparation reagent 2: (1L)
In container, add successively Sodium phosphate, dibasic, KCl, N.F,USP MANNITOL,
Proclin300, adenosine, amino the replacing of 4-mix than woods.
Embodiment 2
Reagent 1: (3L)
MOPS damping fluid 150mmol
toos 6mmol
Triton?x-100 12ml
Sodium Benzoate 72mmol
FeCl3 15mmol
PNP 54u
Xot 90u
Peo 600u
Glycerose 90g
Sodium arseniate 54g
Reagent 2: (1L)
Sodium phosphate, dibasic 40mmol
KCl 15mmol
N.F,USP MANNITOL 25mmol
proclin300 0.5ml
Adenosine 16mmol
4-is amino to be replaced than woods 2mmol
The preparation method is with embodiment 1.
Embodiment 3
Reagent 1: (3L)
HEPES damping fluid 300mmol
toos 6mmol
Triton?x-100 6ml
Sodium Benzoate 9mmol
FeCl3 6mmol
PNP 30u
Xot 60u
Peo 90u
Glycerose 60g
Sodium arseniate 60g
Reagent 2: (1L)
Sodium phosphate, dibasic 50mmol
KCl 2mmol
N.F,USP MANNITOL 20mmol
proclin300 0.3ml
Adenosine 15mmol
4-is amino to be replaced than woods 2mmol
The preparation method is with embodiment 1
Embodiment 4
The test kit of the embodiment of the invention 1 detects effect test:
(1) 0.1M FeCl
3, Sodium Benzoate, Triton x-100 be as the influence of compounding activation to reaction:
Add 0.1M FeCl in the reagent 1
3, Sodium Benzoate, Triton x-100, compare with the reagent that does not add these materials.
Measuring method:
Step 1: input parameter is in automatic biochemistry analyzer: 37 ℃ of temperature, and predominant wavelength 550nm, inferior wavelength 800nm,
R1 225ul.R2 75ul. sample 6ul
Reading point: 0-27 0-10
Step 2: before measuring with R1 and R2 balance to room temperature., put into the reagent storehouse then
Step 3 input scaling ratio: 2100
The compounding activation agent is to the influence of reaction
(2) protective material N.F,USP MANNITOL is to the influence of the enzymic activity in the test kit: add the provide protection of the N.F,USP MANNITOL of 0.1M to enzyme
Above-mentioned detected result shows that the N.F,USP MANNITOL of test kit adding 0.1M of the present invention has very strong provide protection to the enzyme in the reagent; As time goes on; Provide protection meeting to enzyme is more obvious, adds protectant reagent after 12 months and compares with not adding protectant reagent, and remaining enzyme motility rate raises 51%.
The clinical correlation comparison test of embodiment 5 and Mei Liai adenosine deaminase test kit
Present embodiment 1 test kit and Mei Liai adenosine deaminase test kit have been made clinical correlation relatively:
The parallel detection of the clinical sample of getting 30 routine different concns and the commercially available Mei Liai adenosine deaminase test kit that obtains (reagent 1:2*80ml reagent 2:16*10ml) manufacturer (French Mei Liai company)
Use instrument: Olympus 400
Measuring method
Step 1: input parameter is in automatic biochemistry analyzer: 37 ℃ of temperature, and predominant wavelength 550nm, inferior wavelength 800nm,
R1 225ul.R2 75ul. sample 6ul
Reading point: 0-27 0-10
Step 2: before measuring with R1 and R2 balance to room temperature., put into the reagent storehouse then
Step 3 input scaling ratio: 2100
The above parameter of input just can detect on Olympus 400.
Test-results:
According to top clinical comparison data, this reagent is good with the clinical correlation of Mei Liai reagent, does not have difference.Can be used for clinically, import substitutes reduce medical expense.
Claims (3)
1. an enzyme process detects the adenosine deaminase test kit, it is characterized in that, said test kit is made up of with reagent 2 following reagent 1:
Reagent 1:3L
Damping fluid 10-200mmol/L
toos 1-100mmol/L
Triton?x-100 0.01-20ml/L
Sodium Benzoate 20-100mmol/L
FeCl3 1-100mmol/L
PNP 10-20u/L
Xot 30-40u/L
Peo 200-300u/L
Glycerose 10-100g/L
Sodium arseniate 10-100g/L;
Reagent 2:1L
Sodium phosphate, dibasic 10-200mmol/L
KCl 10-500mmol/L
N.F,USP MANNITOL 10-200mmol/L
proclin300 0.01-0.5ml/L
Adenosine 10-200mmol/L
4-is amino to be replaced than woods 1-5mmol/L.
2. detect the preparation method of adenosine deaminase test kit according to the said enzyme process of claim 1, it is characterized in that said reagent 1 damping fluid is selected from TRIS, MOPS or HEPES.
One kind according to claim 1 enzyme process detect the preparation method of adenosine deaminase test kit, it is characterized in that this method comprises the following steps:
(1) preparation reagent 1:3L
(1) in container, is incorporated as the deionized water of total amount 80%;
(2) add successively damping fluid, toos, Triton x-100,
Sodium Benzoate, FeCl3, Glycerose, sodium arseniate;
(3) add enzyme, PNP, Xot, Peo;
(4) adding remainder water at last mixes;
(2) preparation reagent 2:1L
In container, add successively damping fluid, KCl, N.F,USP MANNITOL,
Proclin300, adenosine, amino the replacing of 4-mix than woods.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100042871A CN102586397A (en) | 2011-01-11 | 2011-01-11 | Enzymatic detection adenosine deaminase kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100042871A CN102586397A (en) | 2011-01-11 | 2011-01-11 | Enzymatic detection adenosine deaminase kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102586397A true CN102586397A (en) | 2012-07-18 |
Family
ID=46475620
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011100042871A Pending CN102586397A (en) | 2011-01-11 | 2011-01-11 | Enzymatic detection adenosine deaminase kit |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238847A (en) * | 2015-09-16 | 2016-01-13 | 山东博科生物产业有限公司 | Stable adenosine deaminase reagent high in anti-interference capability and detection method |
| CN105586387A (en) * | 2016-03-11 | 2016-05-18 | 威尚生物技术（合肥）有限公司 | Adenosine deaminase detection kit |
| CN106282309A (en) * | 2016-08-31 | 2017-01-04 | 广东省中医院 | A kind of ADA Adenosine deaminase detection method |
| CN106893759A (en) * | 2015-12-21 | 2017-06-27 | 徐淼 | A kind of adenosine deaminase detection reagent |
| CN108690868A (en) * | 2017-04-10 | 2018-10-23 | 广州市伊川生物科技有限公司 | A kind of adenosine deaminase assay kit and its assay method |
-
2011
- 2011-01-11 CN CN2011100042871A patent/CN102586397A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 周美霞等: "全自动酶偶联法测定腺苷脱氨酶", 《临床检验杂志》 * |
| 潘利琴等: "PNP-XTO-POD偶联单一试剂匀相速率法测定血清5"- NT", 《中国卫生检验杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238847A (en) * | 2015-09-16 | 2016-01-13 | 山东博科生物产业有限公司 | Stable adenosine deaminase reagent high in anti-interference capability and detection method |
| CN106893759A (en) * | 2015-12-21 | 2017-06-27 | 徐淼 | A kind of adenosine deaminase detection reagent |
| CN105586387A (en) * | 2016-03-11 | 2016-05-18 | 威尚生物技术（合肥）有限公司 | Adenosine deaminase detection kit |
| CN106282309A (en) * | 2016-08-31 | 2017-01-04 | 广东省中医院 | A kind of ADA Adenosine deaminase detection method |
| CN108690868A (en) * | 2017-04-10 | 2018-10-23 | 广州市伊川生物科技有限公司 | A kind of adenosine deaminase assay kit and its assay method |
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Application publication date: 20120718 |
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