CN101592667A - A kind of glycosylated hemoglobin detection kit and application thereof - Google Patents
A kind of glycosylated hemoglobin detection kit and application thereof Download PDFInfo
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- CN101592667A CN101592667A CNA2009100653191A CN200910065319A CN101592667A CN 101592667 A CN101592667 A CN 101592667A CN A2009100653191 A CNA2009100653191 A CN A2009100653191A CN 200910065319 A CN200910065319 A CN 200910065319A CN 101592667 A CN101592667 A CN 101592667A
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- glycosylated hemoglobin
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Abstract
The invention belongs to the biologic product technology field, particularly a kind of glycosylated hemoglobin detection kit and application thereof.Described kit comprises confining liquid, stop buffer, substrate solution, also contains following reagent: coating buffer, cleansing solution, antigenic dilution, enzyme-labelled antigen and antibody.Kit of the present invention is formed simple, and is easy to use, the detection sensitivity height, and reliable results, with low cost, efficient is high.The instrumentation degree is low, easy and simple to handle, particularly sample pre-treatments is required simply, is easy to promote.
Description
(1) technical field
The invention belongs to the biologic product technology field, particularly a kind of glycosylated hemoglobin detection kit and application thereof.
(2) background technology
Diabetes have become the third-largest non-infective disease after cardiovascular and cerebrovascular disease and tumour now, also must become and cause one of invalid and dead major reason, glycosylated hemoglobin can be used as the index of the long-term glycemic control of diabetic, and with the generation and the development of chronic complicating diseases of diabetes substantial connection is arranged.Be embodied in 1. in the diabetic danger that the HbA1c level can the predicting cardiovascular disease; 2. in non-diabetic and the normal crowd of HbA1c level, the HbA1c level can be predicted mortality ratio; 3. the patient who has higher concentration HbA1c can obtain benifit from control blood pressure and reduction cholesterol; 4. HbA1c can be used as diabetes or the screening method of sugar tolerance impaired (IGT) patient practicality.
(glycosylated hemoglobin GHb) with HbA1c is representative, and detection method all is to distinguish HbA1c and non-HbA1c according to the difference on physics, chemistry or the immunology to detect glycosylated hemoglobin at present clinically.And these methods all belong to sxemiquantitative, comprise ion-exchange high-voltage liquid phase chromatogram analytic approach, Zeo-karb microtrabeculae chromatography and electrophoresis physical method, suppress method based on the affinity chromatography microtrabeculae method of the principles of chemistry with based on the immune agglutination of immunity principle.Though the method for multiple mensuration glycosylated hemoglobin is arranged, up to the present also there is not a kind of method both can accomplish accurately, be subjected to disturbing factor few, economic again, detection fast.
(3) summary of the invention
The object of the present invention is to provide a kind of glycosylated hemoglobin detection kit, utilize its can realize accurately, economical, fast to the detection of glycosylated hemoglobin.
The technical solution used in the present invention is as follows:
A kind of glycosylated hemoglobin detection kit comprises confining liquid, stop buffer, substrate solution, also contains following reagent:
Coating buffer: the carbonate buffer solution of 0.05mol/L pH8.5-9.6;
Cleansing solution: the PBS that contains the 0.01mol/L pH7.4 of 0.05% (v/v) Tween-20;
The concentration that antigenic dilution: BSA is dissolved in cleansing solution is the solution of 1g/L;
Enzyme-labelled antigen: the antigen of horseradish peroxidase-labeled;
Antibody: mouse-anti people.
The pH of described coating buffer is 9.6.
Also contain the NaN that pH7.3 concentration is 0.05% (wt%) in the described kit
3This reagent can be preserved by the guarantee reagent box in a long time.
Described confining liquid is the PBS that contains the 0.01mol/L pH7.4 of 2% (g/ml) BSA; Described stop buffer is the H of 2mol/L
2SO
4
Described substrate solution can be selected the tmb substrate liquid be made up of A liquid and B liquid:
A liquid:
TMB 20mg
Dimethyl sulfoxide (DMSO) 4ml
C
6H
8O
7·H
2O 1.03g
Distilled water is settled to 100ml;
B liquid:
Na
2HPO
4·12H
2O 1.46g
C
6H
8O
7·H
2O 0.933g
30% hydrogen peroxide, 10 μ l
Deionized water is settled to 100ml, and the pH value is 5.0～5.4.
Other conventional substrate solutions also can use, and A liquid and B liquid equal-volume used when TMB (tetramethyl benzidine) substrate solution used.
Concrete, the prescription of following solution is as follows in the kit:
Coating buffer: get Na
2CO
31.59 gram and NaHCO
32.93 gram is dissolved in the distilled water, and finally adding distil water is settled to 1000ml, stores for future use in 4 ℃.
Cleansing solution: NaCl 8.0 grams, KCl 0.2 gram, Na
2HPO
412H
2O 2.9 grams, KH
2PO
40.2 gram, Tween-20 0.5ml adding distil water are to 1000ml, 4 ℃ store for future use.
Antigenic dilution: BSA 0.1 gram is dissolved in the 100ml cleansing solution.
Confining liquid: BSA 2.0 grams add PBS to 100ml.
Stop buffer: get distilled water 177.8ml, dropwise add 98% concentrated sulphuric acid 22.2ml.
Substrate solution:
A liquid:
TMB 20mg
Dimethyl sulfoxide (DMSO) 4ml
C
6H
8O
7·H
2O 1.03g
Add distilled water and be settled to 100ml;
B liquid:
Na
2HPO
4·12H
2O 1.46g
C
6H
8O
7·H
2O 0.933g
30% hydrogen peroxide, 10 μ l
Deionized water is settled to 100ml, and adjusting the pH value is 5.0～5.4.
The application of described glycosylated hemoglobin detection kit, step is as follows:
(1) bag quilt: with coating buffer coated antibody to 2.5 μ g/ml, 150 μ l/ hole coated elisa plates, 4 ℃ of bags are by 24h;
(2) washing:, 3min/ time, dry with cleansing solution washing 3 times;
(3) sealing: add confining liquid, 300 μ l/ holes, 37 ℃ of incubation 2h;
(4) washing:, 3min/ time, dry with cleansing solution washing 3 times;
(5) reaction: will join in the ELISA Plate hole 37 ℃ of reaction 2h with enzyme-labelled antigen and each 75 μ l mixing of sample to be checked that antigenic dilution dilutes after 40 times;
(6) washing:, 3min/ time, dry with cleansing solution washing 3 times;
(7) add substrate: add substrate solution, 150 μ l/ holes, 37 ℃ of effect 30min;
(8) cessation reaction: add stop bath, 50 μ l/ holes;
(9) measure: survey OD with microplate reader
450nm/ OD
620nmValue.
Invention is started with from the angle of glycosylated hemoglobin structure, amino acid whose variation according to its β chain N end, utilization makes up direct competitive ELISA method at the monoclonal antibody of the specific antigen determinant of glycosylated hemoglobin β chain, be used to detect glycosylated hemoglobin, prepare glycosylated hemoglobin monoclonal antibody enzyme-labelled antigen with horseradish peroxidase (HRP) enzyme that serves as a mark, combine the mouse-anti human IgG with human body glycosylated hemoglobin competitiveness.Amplification with enzymatic reaction shows elementary immunological response.By the composition of rationally determining kit and the concentration that each is formed, realized being beneficial to this kit and detected glycosylated hemoglobin by the ELISA method.
Direct competitive ELISA ratio juris is: specific antibody is adsorbed in solid phase carrier, adds determined antigen and a certain amount of enzyme-labelled antigen, the two is combined with the insolubilized antibody competition, the enzyme-labelled antigen and the determined antigen amount that are incorporated into solid phase after the washing are negative correlation.
The basic process of direct competitive ELISA is as follows:
1. specific antibody and solid phase carrier are connect formation insolubilized antibody, washing, shrouding, washing;
2. Guan Zhongjia to be measured is examined the mixed solution of sample and a certain amount of enzyme-labelled antigen, makes it to react with insolubilized antibody.If examined antigen amount height in the sample, then because the combining of these antigens and insolubilized antibody, the emulative chance that enzyme-labelled antigen combines with insolubilized antibody that accounted for makes the binding capacity of enzyme-labelled antigen and insolubilized antibody few.Only enzyme-added mark antigen in the reference tube, behind the incubation, the amount that can reach fullest that combines of enzyme-labelled antigen and insolubilized antibody, washing;
3. add the substrate colour developing;
4. add cessation reaction liquid;
5. on microplate reader, measure each hole light absorption value with dual wavelength.
In the detection of ELSIA method, the factor that influences sensitivity is a lot.The affinity and specificity, the specific activity of enzyme conjugates and the detected limit value of developer illuminophore absorbance etc. that comprise antibody.The ELISA method of utilizing monoclonal antibody technique to set up, highly sensitive under the rational application conditions of the present invention, reliable results, and also ELISA detection cost is very cheap, and efficient is high.The instrumentation degree is low, easy and simple to handle, particularly sample pre-treatments is required simply, is easy to promote.
Concrete, adopt horseradish peroxidase (HRP) enzyme that serves as a mark, adopt the sodium periodate simplified method coupling HRP and the HbA1c of improvement.Identify that through ultraviolet spectrophotometry the result shows: the mark rate of antigen is 0.565, mole ratio is 1.755, and the mark result meets requirement of experiment.
In the application of kit, with standard items production standard curve, after carry out the mensuration of sample to be measured again, just can obtain testing result.Condition when the making of typical curve, each condition specifically detect together, the curvilinear equation that obtains is y=37.823x-26.833, x is log[HbA1c concentration μ g/ml], y is an inhibiting rate, coefficient R
2=0.9802.Measurement range is 16 μ g/ml-1024 μ g/ml, and sensitivity is 16 μ g/ml; Add the recovery between 96.5-100.3%, average recovery rate is 98.6%; Average coefficient of variation is 2.78% in the plate of kit; The coefficient of variation is 6.22% between plate; Kit can be preserved more than 6 months under 4 ℃.
The present invention has following advantage with respect to prior art:
Kit of the present invention is formed simple, and is easy to use, the detection sensitivity height, and reliable results, with low cost, efficient is high.The instrumentation degree is low, easy and simple to handle, particularly sample pre-treatments is required simply, is easy to promote.
(4) embodiment:
Below with specific embodiment technical scheme of the present invention is described, but protection scope of the present invention is not limited thereto:
Embodiment 1
Glycosylated hemoglobin detection kit, form by following reagent:
Coating buffer: 0.05mol/L pH9.6 carbonate buffer solution;
Cleansing solution: the PBS that contains the 0.01mo l/L pH7.4 of 0.05% Tween-20;
Antigenic dilution: the 100mL cleansing solution that contains 0.1gBSA;
Enzyme-labelled antigen: the antigen of horseradish peroxidase-labeled;
Antibody: mouse-anti people;
Confining liquid: the PBS that contains the 0.01mol/L pH7.4 of 2%BSA;
Stop buffer: 2mol/L H
2SO
4
Substrate solution:
A liquid:
TMB 20mg
Dimethyl sulfoxide (DMSO) 4ml
C
6H
8O
7·H
2O 1.03g
Distilled water is settled to 100ml;
B liquid:
Na
2HPO
4·12H
2O 1.46g
C
6H
8O
7·H
2O 0.933g
30% hydrogen peroxide, 10 μ l
Deionized water is settled to 100ml, and the pH value is 5.2.
Carry out according to following steps during use:
(1) bag quilt: with coating buffer coated antibody to 2.5 μ g/ml, 150 μ l/ hole coated elisa plates, 4 ℃ of bags are by 24h;
(2) washing:, 3min/ time, dry with cleansing solution washing 3 times;
(3) sealing: add confining liquid, 300 μ l/ holes, 37 ℃ of incubation 2h;
(4) washing:, 3min/ time, dry with cleansing solution washing 3 times;
(5) react: respectively get 75 μ l mixings with sample to be checked after enzyme-labelled antigen is diluted 40 times with antigenic dilution and join in the ELISA Plate hole 37 ℃ of reaction 2h;
(6) washing:, 3min/ time, dry with cleansing solution washing 3 times;
(7) add substrate: add substrate solution, 150 μ l/ holes, 37 ℃ of effect 30min;
(8) cessation reaction: add stop bath, 50 μ l/ holes;
(9) measure: survey OD with microplate reader
450nm/ OD
620nmBe worth, obtain the OD of sample to be checked
450nmValue is 0.518; According to calculating the concentration that can obtain glycosylated hemoglobin is 100 μ g/ml.
The stability experiment that utilizes kit to detect
Get reagent at different time and detect, its testing result sees Table 1.As can be seen from Table 1: the B/B of 37 ℃ of 4d
0＜0.7, because of the 24h under 37 ℃ is equivalent to 45d under the room temperature, so kit can be preserved more than 6 months under 4 ℃.
Table 1 kit stability measurement result
Degree of accuracy
The measurement result of error sees Table 2 between the interior sum of errors plate of HbA1c direct competitive ELISA kit plate.By result in the table as can be known, the plate within variance coefficient of HbA1c kit is between 1.852-3.809, and average coefficient of variation is 2.782; The coefficient of variation is between 4.687-8.129 between plate, and average coefficient of variation is 6.219.
The coefficient of variation in the plate of table 2HbA1c kit and between plate
Add the recovery
The results are shown in Table 3.By the result as can be seen, the interpolation recovery of HbA1c direct competitive ELISA method between 96.5%-100.3%, average out to 98.6%.
Table 3 adds recovery experimental result
Claims (6)
1. a glycosylated hemoglobin detection kit comprises confining liquid, stop buffer, substrate solution, it is characterized in that, also contains following reagent:
Coating buffer: the carbonate buffer solution of 0.05mol/L pH 8.5-9.6;
Cleansing solution: the PBS that contains the 0.01mol/L pH7.4 of 0.05% (v/v) Tween-20;
The concentration that antigenic dilution: BSA is dissolved in cleansing solution is the solution of 1g/L;
Enzyme-labelled antigen: the antigen of horseradish peroxidase-labeled;
Antibody: mouse-anti people.
2. glycosylated hemoglobin detection kit as claimed in claim 1 is characterized in that, the pH of described coating buffer is 9.6.
3. glycosylated hemoglobin detection kit as claimed in claim 1 is characterized in that, also contains the pH7.3 mass concentration in the described kit and be 0.05% NaN
3
4. as the described glycosylated hemoglobin detection kit of one of claim 1-3, it is characterized in that confining liquid is the PBS that contains the 0.01mol/L pH7.4 of 2% (g/ml) BSA; Described stop buffer is the H of 2mol/L
2SO
4
5. as the described glycosylated hemoglobin detection kit of one of claim 1-3, it is characterized in that substrate solution is made up of A liquid and B liquid:
A liquid:
TMB 20mg
Dimethyl sulfoxide (DMSO) 4ml
C
6H
8O
7·H
2O 1.03g
Distilled water is settled to 100ml;
B liquid:
Na
2HPO
4·12H
2O 1.46g
C
6H
8O
7·H
2O 0.933g
30% hydrogen peroxide, 10 μ l
Deionized water is settled to 100ml, and the pH value is 5.0～5.4.
6. the application of claim 1 or 2 described glycosylated hemoglobin detection kits is characterized in that step is as follows:
(1) bag quilt: with coating buffer coated antibody to 2.5 μ g/ml, 150 μ l/ hole coated elisa plates, 4 ℃ of bags are by 24h;
(2) washing:, 3min/ time, dry with cleansing solution washing 3 times;
(3) sealing: add confining liquid, 300 μ l/ holes, 37 ℃ of incubation 2h;
(4) washing:, 3min/ time, dry with cleansing solution washing 3 times;
(5) reaction: will dilute 40 times enzyme-labelled antigen and each 75 μ l mixing of sample to be checked join in the ELISA Plate hole, 37 ℃ of reaction 2h with antigenic dilution;
(6) washing:, 3min/ time, dry with cleansing solution washing 3 times;
(7) add substrate: add substrate solution, 150 μ l/ holes, 37 ℃ of effect 30min;
(8) cessation reaction: add stop bath, 50 μ l/ holes;
(9) measure: survey OD with microplate reader
450nm/ OD
620nmValue.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100653191A CN101592667A (en) | 2009-06-30 | 2009-06-30 | A kind of glycosylated hemoglobin detection kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100653191A CN101592667A (en) | 2009-06-30 | 2009-06-30 | A kind of glycosylated hemoglobin detection kit and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101592667A true CN101592667A (en) | 2009-12-02 |
Family
ID=41407434
Family Applications (1)
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|---|---|---|---|
| CNA2009100653191A Pending CN101592667A (en) | 2009-06-30 | 2009-06-30 | A kind of glycosylated hemoglobin detection kit and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197084A (en) * | 2013-03-28 | 2013-07-10 | 山东博科生物产业有限公司 | Stable glycated serum protein detection reagent and application thereof |
| CN103620414A (en) * | 2011-06-28 | 2014-03-05 | 皇家飞利浦有限公司 | Means used for examination of body fluids |
| CN104833663A (en) * | 2012-11-14 | 2015-08-12 | 广西安仁欣生物科技有限公司 | Glycosylated hemoglobin kit based on aptamer fluorescent probe and detection method of same |
-
2009
- 2009-06-30 CN CNA2009100653191A patent/CN101592667A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103620414A (en) * | 2011-06-28 | 2014-03-05 | 皇家飞利浦有限公司 | Means used for examination of body fluids |
| CN104833663A (en) * | 2012-11-14 | 2015-08-12 | 广西安仁欣生物科技有限公司 | Glycosylated hemoglobin kit based on aptamer fluorescent probe and detection method of same |
| CN105044053A (en) * | 2012-11-14 | 2015-11-11 | 广西安仁欣生物科技有限公司 | Glycosylated hemoglobin kit based on aptamer fluorescent probe and detection method thereof |
| CN104833663B (en) * | 2012-11-14 | 2017-08-29 | 广西安仁欣生物科技有限公司 | Glycosylated hemoglobin kit and its detection method based on nucleic acid aptamer fluorescence probe |
| CN105044053B (en) * | 2012-11-14 | 2017-11-03 | 广西安仁欣生物科技有限公司 | Glycosylated hemoglobin kit and its detection method based on nucleic acid aptamer fluorescence probe |
| CN103197084A (en) * | 2013-03-28 | 2013-07-10 | 山东博科生物产业有限公司 | Stable glycated serum protein detection reagent and application thereof |
| CN103197084B (en) * | 2013-03-28 | 2015-04-01 | 山东博科生物产业有限公司 | Stable glycated serum protein detection reagent and application thereof |
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Application publication date: 20091202 |