CN104164473A - Glycated albumin enzymatic detection kit and detection method thereof - Google Patents
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Abstract
The invention relates to a glycated albumin (GA) enzymatic detection kit, which comprises a GA reagent 1 and a GA reagent 2. The GA reagent 1 comprises tris(hydroxymethyl)aminomethane, aminoantipyrine (4-AAP), protease K, calcium acetate (CaAc2), potassium ferrocyanide trihydrate (K4Fe(CN)6.3H2O), copper acetate (CuAc2) and Cholic acid. The GA reagent 2 includes tris(hydroxymethyl)aminomethane, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS), fructosaminase, Triton X-100 and peroxidase. The invention aims to provide the glycated albumin enzymatic detection kit and its detection method with the characteristics of strong specificity, high sensitivity, short time, simple operation mode, and accurate and reliable detection result.
Description
Technical field
The present invention relates to a kind of glycosylated albumin (GA) enzyme process detection kit and detection method thereof.
Background technology
There is the glycosylation of non-enzymatic in the N-end of the glucose in human body and serum protein, wherein 90% with serum protein chain in the 189th Lysine binding, forming high molecular ketoamine structure, be generically and collectively referred to as glycated serum protein, more than 90% is wherein glycosylated albumin.Therefore, glycosylated albumin (Glycated Albumin, GA) can reflect the aggregate level of glycated serum protein.And in the diagnosis and monitoring of diabetes, glycated proteins is significant.Blood sugar monitoring is a very important link in the daily diagnosis and treatment work of diabetes, and good glycemic control can effectively delay generation and the development of the acute and chronic complication of diabetes.In clinical position, will make blood sugar comprehensively up to standard for a long time, the good control of short-term averaging blood sugar is extremely important.Glycosylated albumin (glycatedalbumin, GA) be one of the index of blood sugar monitoring clinically, because the transformation period of albumin in blood is shorter, so can be observed the variation of glycosylated albumin within 2-4 week, for evaluating the recent glycemic control situation of diabetic subject and reflecting that the variation of glucose level has higher clinical value in a short time.But the specificity of the composition of existing mensuration glycated proteins is lower, sensitivity is not high, and the time that obtains detected result is longer, operating method complexity, and detected result is not accurate enough reliable.
Summary of the invention
A kind of high specificity of confession the object of the invention is, highly sensitive, the time that obtains detected result is short, and operating method is easy, glycosylated albumin (GA) concentration results glycosylated albumin (GA) enzyme process detection kit and the detection method thereof accurately and reliably detecting.
Glycosylated albumin enzyme process detection kit of the present invention, comprises GA reagent 1 and GA reagent 2, and GA reagent 1 comprises Tutofusin tris, aminoantipyrene (4-AAP), Proteinase K, lime acetate (CaAc
2), three hydration yellow prussiate of potash (K
4fe (CN)
63H
2o), venus crystals (CuAc
2) and bile acide (Cholic acid); GA reagent 2 comprises Tutofusin tris, N-ethyl-N-TOOS (TOOS), fructosyl amino acid oxidase, triton x-100 (Triton X-100) and peroxidase.
Glycosylated albumin enzyme process detection kit of the present invention, wherein said GA reagent 1 adopts following steps to make:
(1), take 6g Tutofusin tris (TRIS) in container, add suitable quantity of water it dissolved completely, add salt acid for adjusting pH, control pH between 7.95-8.05;
(2), take 0.4g aminoantipyrene (4-AAP), 0.88g lime acetate (CaAc2), 0.02g yellow prussiate of potash (K4Fe (CN) 6) and 0.09g venus crystals (CuAc2), join in the solution that step (1) obtains, fully stir it is dissolved completely;
(3), take 10g bile acide (Cholic acid), join in the solution that step (2) obtains, fully stir it dissolved completely, add hydrochloric acid or sodium hydroxide solution to regulate pH, control pH between 7.95-8.05;
(4), take 3kU Proteinase K, join in the solution that step (3) obtains, fully stir it dissolved completely.
(5), be finally settled to 1000ml with the solution that purified water obtains step (4), filter with 0.2 μ μ m filter, obtain GA reagent 1;
Described GA reagent 2 adopts following steps to make:
(1), take 6g Tutofusin tris (TRIS) in container, add suitable quantity of water it dissolved completely, add salt acid for adjusting pH, control pH between 7.95-8.05;
(2), take 1.47g N-ethyl-N-TOOS (TOOS), join in the solution that step () obtains, fully stir it dissolved completely;
(3), draw 0.5ml triton x-100 (Triton X-100) with pipettor, join in the solution that step (two) obtains, fully stir it is dissolved completely, add hydrochloric acid or sodium hydroxide solution to regulate pH, control pH between 7.95-8.05;
(4), take 100kU fructosyl amino acid oxidase and 200kU peroxidase, join in the solution that step (three) obtains, fully stir it dissolved completely;
(5) in the solution, finally by purified water, step (four) being obtained, be settled to 1000ml, with 0.2 μ μ m filter filtration, obtain GA reagent 2.
The detection method of glycosylated albumin enzyme process detection kit of the present invention, comprises the steps:
1, the GA reagent 1 of 6 μ l samples and 240 μ l is mixed, hatch 5 minutes for 37 DEG C;
2, under 546/700nm wavelength, measure absorbance A 1, wherein predominant wavelength is 546nm, and commplementary wave length is 700nm;
3, the GA reagent 2 that adds 60 μ l mixes, and hatches 5 minutes for 37 DEG C;
4, under 546/700nm wavelength, measure absorbance A 2, calculate Δ A=A1-A2;
5, use calibration object (standard serum), select the quality control product corresponding with calibration object, Quality Control measured value should be within the limits prescribed, adopts following formula to calculate:
Glycosylated albumin of the present invention (GA) enzyme process detection kit and detection method thereof, it comprises GA reagent 1 and GA reagent 2 its test kit, and GA reagent 1 comprises Tutofusin tris, aminoantipyrene (4-AAP), Proteinase K, lime acetate (CaAc
2), three hydration yellow prussiate of potash (K
4fe (CN)
63H
2o), venus crystals (CuAc
2) and bile acide (Cholic acid); GA reagent 2 comprises Tutofusin tris, N-ethyl-N-TOOS (TOOS), fructosyl amino acid oxidase, triton x-100 (Triton X-100) and peroxidase.Experiment shows, glycosylated albumin enzyme process detection kit of the present invention and detection method thereof, have higher sensitivity and specificity, and the time of shorter acquisition detected result and easier operating method.
Below glycosylated albumin of the present invention (GA) enzyme process detection kit and detection method thereof are described in further detail.
Embodiment
The present invention is intended to set up a kind of accurately Fast Measurement glycosylated albumin (Glycated Albumin, GA) method, and research and develop first at home the glycosylated albumin detection kit (enzyme process) of producing commercialization liquid double reagent, detect for clinical labororatory's automatic biochemical analyzer.Cardinal principle of the present invention is to adopt proteolytic enzyme, fructosyl amino acid oxidase (Fructosyl amino acid oxidase, the hydrolysis such as FAOD), catalyzed reaction are measured serum GA content, adopt purpurum bromocresolis colorimetry to carry out quantitatively, obtaining GA per-cent by calculating to albumin simultaneously.Its gordian technique mainly contains FAOD expression, purifying and scale operation; Screening Protease and determination of activity technology; Test kit stability study (containing damping fluid and stablizer screening).The key issue mainly solving has: the stability of raw material calibrating dhdps enzyme, chemical reagent such as () FAOD, proteolytic enzyme; Reagent research system, comprise principle formulating (investigate the raw material that uses whether can realization response principle), experimental formula research (obtains after principle reaction, be optimized for the each index request of reagent), reagent stability evaluation (mainly for life-time service stability study) after optimizing; Product clinical detection suitability.
In the present invention, the fructosyl amino acid oxidase that adopts derives from aspergillus fumigatus (Aspergillus fumigatus), and this enzyme has the reaction of specificity enzymatic equally to free fructosyl amino acid, can reach the object of measuring glycosylated albumin.In addition, about the method for Accurate Measurement serum protein, mainly adopt the purpurum bromocresolis that specificity is higher (BCP) staining.By the combination of two reactions, by can accurately obtaining glycosylated albumin per-cent after measure and calculation.
Glycosylated albumin of the present invention (GA) enzyme process detection kit, comprises GA reagent 1 and GA reagent 2, and GA reagent 1 comprises Tutofusin tris, aminoantipyrene (4-AAP) and Proteinase K; GA reagent 2 comprises Tutofusin tris, N-ethyl-N-TOOS (TOOS), fructosyl amino acid oxidase and peroxidase.
Above-mentioned GA reagent 1 also comprises lime acetate (CaAc
2), three hydration yellow prussiate of potash (K
4fe (CN)
63H
2o), venus crystals (CuAc
2), bile acide (Cholic acid); Described GA reagent 2 also comprises triton x-100 (Triton X-100).
Described GA reagent 1 adopts following steps to make:
(1), take 6g Tutofusin tris (TRIS) in container, add suitable quantity of water it dissolved completely, add salt acid for adjusting pH, control pH between 7.95-8.05;
(2), take 0.4g aminoantipyrene (4-AAP), 0.88g lime acetate (CaAc2), 0.02g yellow prussiate of potash (K4Fe (CN) 6) and 0.09g venus crystals (CuAc2), join in the solution that step (1) obtains, fully stir it is dissolved completely;
(3), take 10g bile acide (Cholic acid), join in the solution that step (2) obtains, fully stir it dissolved completely, add hydrochloric acid or sodium hydroxide solution to regulate pH, control pH between 7.95-8.05;
(4), take 3kU Proteinase K, join in the solution that step (3) obtains, fully stir it dissolved completely.
(5), be finally settled to 1000ml with the solution that purified water obtains step (4), filter with 0.2 μ μ m filter, obtain GA reagent 1;
Described GA reagent 2 adopts following steps to make:
(1), take 6g Tutofusin tris (TRIS) in container, add suitable quantity of water it dissolved completely, add salt acid for adjusting pH, control pH between 7.95-8.05;
(2), take 1.47gN-ethyl-N-TOOS (TOOS), join in the solution that step () obtains, fully stir it dissolved completely;
(3), draw 0.5ml triton x-100 (Triton X-100) with pipettor, join in the solution that step (two) obtains, fully stir it is dissolved completely, add hydrochloric acid or sodium hydroxide solution to regulate pH, control pH between 7.95-8.05;
(4), take 100kU fructosyl amino acid oxidase and 200kU peroxidase, join in the solution that step (three) obtains, fully stir it dissolved completely;
(5) in the solution, finally by purified water, step (four) being obtained, be settled to 1000ml, with 0.2 μ μ m filter filtration, obtain GA reagent 2.
The detection method of glycosylated albumin enzyme process detection kit, is characterized in that: comprise the steps:
1, the GA reagent 1 of 6 μ l samples and 240 μ l is mixed, hatch 5 minutes for 37 DEG C;
2, under 546/700nm wavelength, measure absorbance A 1, wherein predominant wavelength is 546nm, and commplementary wave length is 700nm;
3, the GA reagent 2 that adds 60 μ l mixes, and hatches 5 minutes for 37 DEG C;
4, under 546/700nm wavelength, measure absorbance A 2, calculate Δ A=A1-A2;
5, use calibration object (standard serum), select the quality control product corresponding with calibration object, Quality Control measured value should be within the limits prescribed, adopts following formula to calculate:
Had above-mentioned glycosylated albumin (GA) concentration, also needed to obtain albumin (ALB) concentration by other experiments, utilized above formula to draw albumin (ALB) concentration, recycling following formula draws:
The significant parameter of above-mentioned detection test is as follows:
Method: end-point method
Temperature: 37 DEG C
Predominant wavelength: 546nm
Commplementary wave length: 700nm
Sample size: 6.0 μ l
GA reagent 1:240 μ l
GA reagent 2:60 μ l
The Direction of Reaction: forward
The preincubate time: 5 minutes
Minute: 5 minutes
Calibrating mode: 2 calibrations.
Claims (3)
1. glycosylated albumin enzyme process detection kit, is characterized in that: comprise GA reagent 1 and GA reagent 2, GA reagent 1 comprises Tutofusin tris, aminoantipyrene (4-AAP), Proteinase K, lime acetate (CaAc
2), three hydration yellow prussiate of potash (K
4fe (CN)
63H
2o), venus crystals (CuAc
2) and bile acide (Cholic acid); GA reagent 2 comprises Tutofusin tris, N-ethyl-N-TOOS (TOOS), fructosyl amino acid oxidase, triton x-100 (Triton X-100) and peroxidase.
2. according to glycosylated albumin enzyme process detection kit claimed in claim 1, it is characterized in that:
Described GA reagent 1 adopts following steps to make:
(1), take 6g Tutofusin tris (TRIS) in container, add suitable quantity of water it dissolved completely, add salt acid for adjusting pH, control pH between 7.95-8.05;
(2), take 0.4g aminoantipyrene (4-AAP), 0.88g lime acetate (GaAc2), 0.02g yellow prussiate of potash (K4Fe (CN) 6) and 0.09g venus crystals (CuAc2), join in the solution that step (1) obtains, fully stir it is dissolved completely;
(3), take 10g bile acide (Cholic acid), join in the solution that step (2) obtains, fully stir it dissolved completely, add hydrochloric acid or sodium hydroxide solution to regulate pH, control pH between 7.95-8.05;
(4), take 3kU Proteinase K, join in the solution that step (3) obtains, fully stir it dissolved completely.
(5), be finally settled to 1000ml with the solution that purified water obtains step (4), filter with 0.2 μ μ m filter, obtain GA reagent 1;
Described GA reagent 2 adopts following steps to make:
(1), take 6g Tutofusin tris (TRIS) in container, add suitable quantity of water it dissolved completely, add salt acid for adjusting pH, control pH between 7.95-8.05;
(2), take 1.47gN-ethyl-N-TOOS (TOOS), join in the solution that step () obtains, fully stir it dissolved completely;
(3), draw 0.5ml triton x-100 (Triton X-100) with pipettor, join in the solution that step (two) obtains, fully stir it is dissolved completely, add hydrochloric acid or sodium hydroxide solution to regulate pH, control pH between 7.95-8.05;
(4), take 100kU fructosyl amino acid oxidase and 200kU peroxidase, join in the solution that step (three) obtains, fully stir it dissolved completely;
(5) in the solution, finally by purified water, step (four) being obtained, be settled to 1000ml, with 0.2 μ μ m filter filtration, obtain GA reagent 2.
3. the detection method of glycosylated albumin enzyme process detection kit, is characterized in that: comprise the steps:
1, the GA reagent 1 of 6 μ l samples and 240 μ l is mixed, hatch 5 minutes for 37 DEG C;
2, under 546/700nm wavelength, measure absorbance A 1, wherein predominant wavelength is 546nm, and commplementary wave length is 700nm;
3, the GA reagent 2 that adds 60 μ l mixes, and hatches 5 minutes for 37 DEG C;
4, under 546/700nm wavelength, measure absorbance A 2, calculate Δ A=A1-A2;
5, use calibration object (standard serum), select the quality control product corresponding with calibration object, Quality Control measured value should be within the limits prescribed, adopts following formula to calculate:
。
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673878A (en) * | 2015-03-13 | 2015-06-03 | 温州医科大学 | Kit for measuring concentration ratio of glycated albumin and albumin by virtue of single system |
| CN104990879A (en) * | 2015-07-02 | 2015-10-21 | 董静平 | Serum glycated albumin content measuring method and measuring kit |
| CN108070634A (en) * | 2017-11-24 | 2018-05-25 | 宁波美康保生生物医学工程有限公司 | Three combined detection reagent disks of diabetes |
| CN108254241A (en) * | 2018-02-06 | 2018-07-06 | 三诺生物传感股份有限公司 | A kind of glycosylated albumin blood sample treatment fluid and its application |
| CN109477839A (en) * | 2016-07-28 | 2019-03-15 | 旭化成制药株式会社 | The measuring method of glycosylated albumin |
| CN109613266A (en) * | 2018-12-30 | 2019-04-12 | 吉林大学 | It is a kind of detection glycosylated albumin and its concentration method, detection glycated amino acid oxidizing ferment -one amine oxidase and its concentration method |
| CN109991426A (en) * | 2019-04-03 | 2019-07-09 | 深圳市安帝宝科技有限公司 | A kind of glycated albumin detection kit |
| JP2021007345A (en) * | 2019-07-01 | 2021-01-28 | 旭化成ファーマ株式会社 | Glycated protein measurement reagent containing dimethyl sulfoxide, method for measuring glycated protein, method for storing glycated protein assay reagent, and method for stabilizing glycated protein measurement reagent |
| CN114158267A (en) * | 2019-07-01 | 2022-03-08 | 旭化成制药株式会社 | Glycated protein measurement reagent containing protease stabilizer for increasing oxidation-reduction potential of ferrocyanide, method for measuring glycated protein, method for storing glycated protein measurement reagent, and method for stabilizing glycated protein measurement reagent |
| CN114480565A (en) * | 2021-12-24 | 2022-05-13 | 桂林优利特医疗电子有限公司 | Stable high-sensitivity glycated albumin determination kit with strong anti-interference capability |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673878A (en) * | 2015-03-13 | 2015-06-03 | 温州医科大学 | Kit for measuring concentration ratio of glycated albumin and albumin by virtue of single system |
| CN104990879A (en) * | 2015-07-02 | 2015-10-21 | 董静平 | Serum glycated albumin content measuring method and measuring kit |
| CN109477839A (en) * | 2016-07-28 | 2019-03-15 | 旭化成制药株式会社 | The measuring method of glycosylated albumin |
| CN109477839B (en) * | 2016-07-28 | 2022-06-14 | 旭化成制药株式会社 | Method for measuring glycated albumin |
| CN108070634B (en) * | 2017-11-24 | 2021-04-06 | 宁波美康保生生物医学工程有限公司 | Three-item combined detection reagent tray for diabetes |
| CN108070634A (en) * | 2017-11-24 | 2018-05-25 | 宁波美康保生生物医学工程有限公司 | Three combined detection reagent disks of diabetes |
| CN108254241A (en) * | 2018-02-06 | 2018-07-06 | 三诺生物传感股份有限公司 | A kind of glycosylated albumin blood sample treatment fluid and its application |
| CN109613266A (en) * | 2018-12-30 | 2019-04-12 | 吉林大学 | It is a kind of detection glycosylated albumin and its concentration method, detection glycated amino acid oxidizing ferment -one amine oxidase and its concentration method |
| CN109991426A (en) * | 2019-04-03 | 2019-07-09 | 深圳市安帝宝科技有限公司 | A kind of glycated albumin detection kit |
| CN114158267A (en) * | 2019-07-01 | 2022-03-08 | 旭化成制药株式会社 | Glycated protein measurement reagent containing protease stabilizer for increasing oxidation-reduction potential of ferrocyanide, method for measuring glycated protein, method for storing glycated protein measurement reagent, and method for stabilizing glycated protein measurement reagent |
| JP2021007345A (en) * | 2019-07-01 | 2021-01-28 | 旭化成ファーマ株式会社 | Glycated protein measurement reagent containing dimethyl sulfoxide, method for measuring glycated protein, method for storing glycated protein assay reagent, and method for stabilizing glycated protein measurement reagent |
| JP7328808B2 (en) | 2019-07-01 | 2023-08-17 | 旭化成ファーマ株式会社 | Reagent for measuring glycated protein containing dimethylsulfoxide, method for measuring glycated protein, method for storing reagent for measuring glycated protein, and method for stabilizing reagent for measuring glycated protein |
| CN114480565A (en) * | 2021-12-24 | 2022-05-13 | 桂林优利特医疗电子有限公司 | Stable high-sensitivity glycated albumin determination kit with strong anti-interference capability |
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