CN102747133A - Adenosine deaminase (ADA) detection reagent kit and preparation method thereof - Google Patents
Adenosine deaminase (ADA) detection reagent kit and preparation method thereof Download PDFInfo
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- CN102747133A CN102747133A CN2012102612658A CN201210261265A CN102747133A CN 102747133 A CN102747133 A CN 102747133A CN 2012102612658 A CN2012102612658 A CN 2012102612658A CN 201210261265 A CN201210261265 A CN 201210261265A CN 102747133 A CN102747133 A CN 102747133A
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Abstract
The invention discloses a detection reagent kit for detecting human ADA. A reagent kit body contains a glycine buffer solution, a color-developing agent prepared with 3-methyl-N,N-dipropyl sodium sulfonate aniline, purine nucleoside phosphorylase, xanthine oxidase, peroxidase, adenosine and a 4-aminoantipyrine solution. A sample and a reagent are mixed in a certain volume proportion and subjected to a series of reactions, then reactants are placed under a semi-automatic/full-automatic biochemical analyzer, and the change rate of the absorbency at a position of 550nm dominant wavelength is detected, so that the concentration of the ADA is measured and calculated. The reagent kit has the advantages of being accurate, stable and convenient.
Description
Technical field
The invention belongs to the biological medicine technology field, relate in particular to a kind of hADA's of detection detection kit.
Background technology
Adenosine deaminase (ADA) is the enzyme of keying action in the purine nucleoside catabolic process, and it can be degraded to inosine by the catalysis adenosine, and after nucleoside phosphorylase is catalyzed into xanthoglobulin, finally is oxidized to the meta-bolites uric acid.Adenosine deaminase has three kinds of isozymes, is respectively adenosine deaminase 1, adenosine deaminase 1+cp and adenosine deaminase 2.
Adenosine deaminase distribution in animal body is the highest with content in caecum, mucous membrane of small intestine and the spleen; Locate also to have discovery at fibrocyte, amniocyte, liver, kidney, lung, Skelettmuskel etc.; ADA mainly is present in the hemocyte in the blood, and like red corpuscle, lymphocyte and granulocyte, its activity is about 40～70 times in the serum; Activity of adenosine deaminase in the tissue can keep a week under freezing condition, but in tissue homogenate the cryopreservation several months long.
Activity of adenosine deaminase is the sensitive indicator of reflection liver injury.Chronic hepatitis, liver cirrhosis and hepatocellular carcinoma patients serum activity of adenosine deaminase significantly raise.Its positive rate reaches 85%～90%, so adenosine deaminase activity determination can be used as the screening index of chronic hepatopathy.The decompensated liver cirrhosis activity of adenosine deaminase is apparently higher than compensatory cirrhosis, thereby can judge the degree of chronic hepatopathy.In addition, the chronic active hepatitis activity of adenosine deaminase is apparently higher than chronic persistent hepatitis, so can be used for the differential diagnosis of the two.
The ADA detection method has formed two kinds of main detection methods of ADA detection kit in the market through repeatedly improving, and comprises continuous detecting method and enzyme coupling method.
It is that ADA produces Trophicardyl and ammonia with the adenosine deamination that ADA the earliest detects principle.Low concentration of substrate does not reach the saturated requirement of substrate, causes the active detection of ADA distortion.Therefore, this method is not suitable for clinical application.The principle of the s-generation ADA of development detection subsequently is the hydrolysis of adenosine deaminase catalysis adenosine, produces inosine and ammonia.The required reagent of this method is prone to preparation, and instrument is simple, but sensitivity is low, is subject to exogenous NH3 influence, and blank too high, it is active directly to measure red corpuscle ADA.
Same reason, the reaction of ADA coupling glutamate dehydrogenase: the speed through measuring the decline of the 340nm NADPH of place absorbancy is calculated the ADA activity.This method is also disturbed because of serum contains ammonia, and too high NADPH causes non-specific oxidation and do not have preconceived plan in the test macro.
Summary of the invention
The detection kit that the purpose of this invention is to provide a kind of detection hADA (ADA).Accuracy is strong as a result, reagent stability good for this test kit, easy to use, be convenient to large-scale promotion.
Another object of the present invention provides the production preparation and the method for use of above-mentioned detection kit.
For realizing above-mentioned purpose, test kit provided by the invention consists of:
1. glycine buffer, 2. developer, 3. purine nucleoside phosphorylase, 4. XOD, 5. px, 6. adenosine, 7.4-aminoantipyrene solution.。
It is following that the preparation method of mentioned reagent box provided by the invention and behaviour do step:
(a) prepare reagent in following ratio:
Glycine buffer 80mmol/L
Developer 10mmol/L
Purine nucleoside phosphorylase 0.5-1KU/L
XOD 0.8-1KU/L
Px 0.6-1KU/L
Adenosine 30mmol/L
4-aminoantipyrene solution 10mmol/L
(b) reagent is mixed with sample to be tested by a certain percentage, make its complete reaction;
(c) measure the absorbancy changing value with half/automatic clinical chemistry analyzer;
(d) calculate the concentration of adenosine deaminase in the sample according to the absorbancy changing value.
The uniqueness of test kit of the present invention is, developer is for containing 3-methyl-N, the phosphate buffered saline buffer of N-dipropyl sodium sulfonate aniline, and 4-aminoantipyrene solution is the chloroformic solution that contains the 4-aminoantipyrene.Through after the series reaction, can form quinones, have color developing effect good, be swift in response, stable a bit.
Embodiment
Following embodiment is for the more detailed the present invention of explanation, is confined to this but should not be construed as the present invention.
Embodiment 1:
Test kit of the present invention can be double reagent for example, wherein:
Reagent 1:
Glycine buffer 80mmol/L
Developer 10mmol/L
Purine nucleoside phosphorylase 0.5KU/L
XOD 0.8KU/L
Px 0.6KU/L
Reagent 2:
Adenosine 30mmol/L
4-aminoantipyrene solution 10mmol/L
Wherein:
(1) glycine buffer is the 35mM glycocoll, 0.05% tween 20,10mM EDTA Disodium, 0.05% Thiomersalate, 0.4M NaCl, pH6.5-10;
(2) developer is for containing 20mM3-methyl-N, the phosphate buffered saline buffer of N-dipropyl sodium sulfonate aniline;
(3) 4-aminoantipyrene solution is the chloroformic solution that contains 25mM 4-aminoantipyrene.
Embodiment 2: the test kit method of use
1. reagent is prepared, and reagent can be liquid double reagent, and uncork i.e. usefulness, wherein:
Reagent 1:
Glycine buffer 80mmol/L
Developer 10mmol/L
Purine nucleoside phosphorylase 0.5KU/L
XOD 0.8KU/L
Px 0.6KU/L
Reagent 2:
Adenosine 30mmol/L
4-aminoantipyrene solution 10mmol/L
2. the full-automatic biochemical instrument parameter is provided with
(a) detected temperatures: 37 ℃
(b) detect wavelength: predominant wavelength is 550nm; Commplementary wave length is 700 nm;
(c) reaction times: 10 minutes, wherein, 5 minutes incubation time, 3 minutes reaction times, 2 minutes detection times.
3. detect step (all in automatic clinical chemistry analyzer, accomplishing)
(a) get 180 μ L reagent 1 and 5 μ L serum sample mixings.
(b) with the solution behind the mixing 37 ℃ of incubations 5 minutes.
(c) add 60 μ L reagent 2, react after 3 minutes, the absorbancy that under the 550nm condition, detects 2 minutes changes.
4. the concentration that calculates adenosine deaminase that changes through absorbancy.
The advantage that the present invention has:
Accuracy is strong as a result, reagent stability good for this test kit, easy to use, be convenient to large-scale promotion.Required detecting instrument (Biochemical Analyzer) generally uses at various big hospital and inspection center.
Claims (8)
1. adenosine deaminase detection kit, it consists of:
Glycine buffer, developer, purine nucleoside phosphorylase, XOD, px, adenosine, 4-aminoantipyrene solution.
2. the test kit of claim 1 is characterized in that:
Glycine buffer is the 15-45mM glycocoll, 0.05% tween 20,10mM EDTA Disodium, 0.05% Thiomersalate, 0.4M NaCl, pH6.5-10;
Developer is for containing 20mM 3-methyl-N, the phosphate buffered saline buffer of N-dipropyl sodium sulfonate aniline;
4-aminoantipyrene solution is the chloroformic solution that contains 20-30mM 4-aminoantipyrene.
3. the preparation of mentioned reagent box and working method, key step is:
(a) prepare reagent in following ratio:
Glycine buffer 80mmol/L
Developer 10mmol/L
Purine nucleoside phosphorylase 0.5-1KU/L
XOD 0.8-1KU/L
Px 0.6-1KU/L
Adenosine 30mmol/L
4-aminoantipyrene solution 10mmol/L
(b) reagent is mixed with sample to be tested by a certain percentage, make its complete reaction;
(c) measure the absorbancy changing value with half/automatic clinical chemistry analyzer;
(d) calculate the concentration of adenosine deaminase in the sample according to the absorbancy changing value.
4. the method for claim 3 is characterized in that, sample and ratio of reagents should be controlled at 1:45 between the 1:60.
5. the method for claim 3 is characterized in that, temperature of reaction is 37 (± 1) ℃.
6. the method for claim 3 is characterized in that, the reaction times is 10-15 minute.
7. the method for claim 3 is characterized in that, the predominant wavelength of using half/automatic clinical chemistry analyzer to detect is 550nm, and commplementary wave length is 700 nm.
8. the method for claim 3 is characterized in that, the configurable one-tenth single reagent of reagent, double reagent or three reagent.
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| CN201210261265.8A CN102747133B (en) | 2012-07-26 | 2012-07-26 | Adenosine deaminase (ADA) detection reagent kit and preparation method thereof |
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| CN102747133B CN102747133B (en) | 2014-06-25 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586387A (en) * | 2016-03-11 | 2016-05-18 | 威尚生物技术（合肥）有限公司 | Adenosine deaminase detection kit |
| CN106893759A (en) * | 2015-12-21 | 2017-06-27 | 徐淼 | A kind of adenosine deaminase detection reagent |
| CN108949900A (en) * | 2018-07-27 | 2018-12-07 | 金华市强盛生物科技有限公司 | One kind can continuously monitor adenosine deaminase detection kit and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709324A (en) * | 2009-12-24 | 2010-05-19 | 张闻 | Method for calibrating and measuring activity of adenosine deaminase, kit matched and use thereof |
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2012
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709324A (en) * | 2009-12-24 | 2010-05-19 | 张闻 | Method for calibrating and measuring activity of adenosine deaminase, kit matched and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| 赵阳: "临床化学体外诊断试剂（盒）产品命名现状及规范化研究", 《首都医药》, no. 6, 30 June 2012 (2012-06-30), pages 5 - 7 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108949900A (en) * | 2018-07-27 | 2018-12-07 | 金华市强盛生物科技有限公司 | One kind can continuously monitor adenosine deaminase detection kit and preparation method thereof |
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| CN102747133B (en) | 2014-06-25 |
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Address after: 102200 the 3 floor of the 4 production building, No. 5, Chao Qian Road, Changping District science and Technology Park, Beijing. Patentee after: Lepu (Beijing) Diagnostic Technology Co.,Ltd. Address before: 100083 the 3 floor of the 4 production building, No. 5, Chao Qian Road, Changping District science and Technology Park, Beijing. Patentee before: Beijing Hongji Polytron Technologies Inc. and biological Address after: 100083 the 3 floor of the 4 production building, No. 5, Chao Qian Road, Changping District science and Technology Park, Beijing. Patentee after: Beijing Hongji Polytron Technologies Inc. and biological Address before: 100083 room 1801, Xue Hun 16, Xue Qing Road, Haidian District, Beijing. Patentee before: BEIJING ENJIHE BIOTECHNOLOGY Co.,Ltd. |