CN102175670B - Method for detecting 1,5-dehydration glucitol in blood and kit - Google Patents
Method for detecting 1,5-dehydration glucitol in blood and kit Download PDFInfo
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Abstract
The invention relates to a method for detecting 1,5-dehydration glucitol in blood and a kit, and provides a new method for detecting the content of the 1,5-dehydration glucitol in the blood, which comprises the following steps of: catalyzing the 1,5-dehydration glucitol by using pyranose oxidase to generate 1,5-dehydration fructose and H2O2; generating quinine compounds by using 4-aminoantipyrine (4-AAP), 3-hydroxy-2,4,6-trihydroxybenzoic acid (HTIB) and H2O2 under the catalytic action of horse radish peroxidase; and determining the level of the 1,5-dehydration glucitol in the blood by colorimetric analysis. The invention also provides the kit for the method. The method and the kit are easy and convenient to operate safely, long in stable time and high in interference resistance, specificity and sensitivity, the interference of multiple glucoses in blood samples can be eliminated quickly, and the reliable foundation can be provided for the diagnosis and treatment of diabetes.
Description
Technical field
The present invention relates to biochemical technology and biochemical test technology, especially for 1,5-AG in the external quantitative measurement blood (1,5-AG) detection method of content and kit.
Background technology
Diabetes spp is in common clinical, frequently-occurring disease, reason is that hypoinsulinism causes, 1,5-AG in the blood (1,5-AG) be significant negative correlation with glucose (GLU), glycosylated hemoglobin (HbAlc), fructosamine (FMN).Have data to show, plasma glucose (GLU)＞8.3mmol/L person, none example with high-caliber blood plasma 1,5-AG (1,5-AG).
1,5-AG (1,5-AG) can be used as the sensitivity specific index of diabetic's early diagnosis, also can be used as treating diabetes with more after the evaluation index of observing.Existing diabetes diagnosis index has obvious deficiency, and fasting plasma glucose is insensitive, and its decision level is difficult to final conclusion, and is especially higher for type ii diabetes patient rate of missed diagnosis; Though oral glucose tolerance experiment capable of dynamic Monitoring Blood Glucose changes, complex operation for using danger larger in the observation in treatment, after more of patient diagnosed, and easily brings out the hyperglycaemia crisis; Glycosylated hemoglobin (HbAlc) belongs to semi-quantitative analysis although be a kind of early diagnosis index, can not satisfy clinical requirement.
1,5-AG (1,5-AG) all present significant negative correlation, the order of severity significant correlation of the minimizing degree of 1,5-AG and diabetes in the blood with existing diabetes index.1,5-dewatered grape sugar alcohol (1, that cycle of reflection diabetes change of illness state is different from the significant difference of other indexs 5-AG), blood sugar (GLU) reflection patient is blood glucose condition at that time, the long-run average of blood sugar in fructosamine (FMN) the reaction patient two weeks, nearest about 3 months blood sugar average level of glycosylated hemoglobin (HbAlc) reflection patient, and 1,5-dewatered grape sugar alcohol (1,5-AG) can reflect within the next few days average blood sugar level to 1 week.
Present main employing holoenzyme method mensuration 1,5-AG both at home and abroad (1,5-AG), mainly contain two kinds of methods of oxidation enzyme process and dehydrogenation enzyme process.These two kinds of methods all need a series of enzyme to participate in eliminating the glucose interference and the generation coloring matter detects.At present major defect is the raw material sources difficulties, diaphorase (DI), EC1.1.99.13 (1,5-AG-HK) all higher with pyranose oxidase (PROD) (oxidase method) price, and the source difficulty.Mostly be greatly in the market powdered reagent (available reagent less stable), detect interference such as being subject to easily endogenous material such as glucose, cholerythrin, ascorbic acid, blood fat, specificity remains further to be improved.
Summary of the invention
The invention provides the method for 1,5-AG in a kind of new mensuration blood, in the method, by pyranose oxidase (PROD) catalysis 1,5-AG (1,5-AG) generate 1,5-anhydrofructose and H
2O
24-AAP (4-AAP), 3-hydroxyl-2,4,6-trihydroxybenzoic acid (HTIB) and H
2O
2Under the catalytic action of horseradish peroxidase (HRP), generate quinones (redness); Utilize the colorimetric analysis principle can draw 1,5-AG in the blood (1,5-AG) level.
Therefore, one aspect of the present invention relates to a kind of method of measuring 1,5-dehydration glucitol in blood content, and described method is by following reaction assay 1,5-dehydration glucitol in blood content:
Wherein, utilize the glucose in glucose oxidase and the hydrogen peroxidase removing blood, utilize subsequently pyranose oxidase catalysis 1,5-dewatered grape sugar alcohol produces hydrogen peroxide, chromogen and hydrogen peroxide produce coloured substrate under the catalytic action of horseradish peroxidase, carry out colorimetric analysis and determine thus the content of 1,5-dehydration glucitol in blood.
The inventor finds using method of the present invention to measure blood 1, during 5-dewatered grape sugar alcohol content, can further select one or more the combination in the following step: the combination that can add potassium ferrocyanide and surfactant is eliminated in the sample high cholerythrin to the impact of sample measured value; Also can be by triglyceride in the adding surfactant elimination sample on the impact of sample measured value; Also can be by high ascorbic acid in the adding ascorbic acid oxidase elimination sample on the impact of sample.
The present invention provides a kind of mensuration blood 1 on the other hand, the kit of 5-dewatered grape sugar alcohol content, comprise reagent I and reagent II two parts, wherein reagent I contains glucose oxidase, hydrogen peroxidase, ascorbic acid oxidase, surfactant, potassium ferrocyanide, damping fluid, antiseptic, stabilizing agent; Reagent II contains pyranose oxidase, 4-AAP, horseradish peroxidase, 3-hydroxyl-2,4,6-trihydroxybenzoic acid, damping fluid, antiseptic, stabilizing agent.
In the kit provided by the invention, the content of each component can be in every liter of reagent I solution:
Glucose oxidase (GOD) 1-50KU
Hydrogen peroxidase (CAT) 1-20KU
Ascorbic acid oxidase (ASO) 1-20KU
Surfactant 0.01-10%
Potassium ferrocyanide 0.1-10000umol
Phosphate buffer (PBS) 1-500mmol
Stabilizing agent 0.01-10%
Antiseptic 0.01-10%;
The content of each component can be among every liter of reagent II:
Pyranose oxidase (PROD) 1-500KU
4-AAP (4-AAP) 0.1-5mmol
Horseradish peroxidase (HRP) 1-20KU
3-hydroxyl-2,4,6-trihydroxybenzoic acid (HTIB) 0.1-10mmol
Phosphate buffer (PBS) 1-500mmol
Stabilizing agent 0.01-10%
Antiseptic 0.01-10%.
In the kit provided by the invention, the content of each component can be in every liter of reagent I solution:
Glucose oxidase (GOD) 15KU
Hydrogen peroxidase (CAT) 2KU
Ascorbic acid oxidase (ASO) 10KU
Surfactant 0.05%
Potassium ferrocyanide 100umol
Phosphate buffer (PBS) 100mmol
Stabilizing agent 0.05%
Antiseptic 0.05%;
The content of each component can be among every liter of reagent II:
Pyranose oxidase (PROD) 200KU
4-AAP (4-AAP) 5mmol
Horseradish peroxidase (HRP) 10KU
3-hydroxyl-2,4,6-trihydroxybenzoic acid (HTIB) 5mmol
Phosphate buffer (PBS) 400mmol
Stabilizing agent 0.05%
Antiseptic 0.05%.
The surfactant of use can be in the present invention, for example one or more in triton x-100, B66, polysorbas20, neopelex, betaine, the cetyl trimethyl ammonium bromide.
Stabilizing agent and the antiseptic of use can be in the present invention, for example one or more in Sodium azide, nitrine lithium, PC300, Sodium Benzoate, potassium sorbate, propylparaben, polyglycol, polyvinyl alcohol (PVA), the disodium ethylene diamine tetraacetate.
Use detection reagent provided by the invention to measure in the blood 1,5-dewatered grape sugar alcohol (1,5-AG) content, has simple to operate, safety, have the characteristics such as specificity is good, sensitivity is high, reagent I and sample to be tested effect can be removed the interference of the polyhydroxy-alcohols such as glucose in blood after 5 minutes, Effective Raise the reagent of the present invention specificity and the accuracy that detect.Use reagent of the present invention to detect serum sample and can avoid sample haemolysis, piarhemia, the impact of jaundice on measuring, improved the specificity that detects.The degree of accuracy of reagent of the present invention in a few days CV is 0.5-2%, and to diabetes, especially the diagnosis and treatment of potential type case provide reliable diagnosis basis, is that individual event mensuration is diagnosable inspection method with judging after I, type ii diabetes are healed.
Description of drawings
Fig. 1: the correlativity of Self-made reagent and contrast agents measurement result.
Fig. 2: the anti-cholerythrin effect of Self-made reagent and contrast agents relatively.
Fig. 3: the anti-triglyceride effect of Self-made reagent and contrast agents relatively.
Fig. 4: Self-made reagent and contrast agents stability are relatively.
Embodiment
1. preparation detects and uses reagent
Get:
Glucose oxidase (GOD) 15KU
Hydrogen peroxidase (CAT) 2KU
Ascorbic acid oxidase (ASO) 10KU
Cetyl trimethyl ammonium bromide 1g
Potassium ferrocyanide 100umol
Phosphate buffer (PBS) 100mmol
Polyglycol 0.5g
Sodium Benzoate 0.5g
Be mixed with 1 liter of solution generate a reagent I;
Get:
Pyranose oxidase (PROD) 200KU
4-AAP (4-AAP) 5mmol
Horseradish peroxidase (HRP) 10KU
3-hydroxyl-2,4,6-trihydroxybenzoic acid (HTIB) 5mmol
Phosphate buffer (PBS) 400mmol
Polyglycol 0.5g
Sodium azide 0.5g
Be mixed with 1L solution generate a reagent II.
2. preparation 1,5-AG calibration object: claim aequum sterling (purchase of Sigma company) to be dissolved in the pure water, and add 0.1% Sodium azide and cook antiseptic that sealing stores for future use.
3. preparation 1,5-AG quality-control product: claim aequum sterling (purchase of Sigma company) to be dissolved in the pure water, and add 0.1% Sodium azide and cook antiseptic that sealing stores for future use.
4. detect operation
37 ℃ of reactions of mixing 10 minutes with the pure water zeroing, are measured the absorbance of each pipe.
5. calculate and normal value
According to this laboratory condition, optional mensuration by hand or automatic clinical chemistry analyzer, revise experiment parameter and make reagent I: reagent II: sample=30: 10: 1, haemolysis on experimental result without impact, jaundice, piarhemia on experimental result without impact.
6. the preparation of contrast agents (reagent that uses in the prior art):
Pyruvate kinase 3KU
Glucokinase 4KU
Ascorbic acid oxidase 10KU
Atriphos 1.0mmol
Phosphoenolpyruvic acid 4.0mmol
4-AAP 1.5mmol
Magnesium chloride 7.5mmol
Potassium chloride 5.0mmol
Acetate buffer 200mmol
Be mixed with 1 liter of solution generate a reagent I;
Get:
Pyranose oxidase 100KU
Horseradish peroxidase 10KU
Phenol 22mmol
Acetate buffer 200mmol
Be mixed with 1L solution generate a reagent II.
Use formulated reagent of the present invention (Self-made reagent) and prior art reagent (contrast agents) to measure 1,5-AG results relevance good (Fig. 1), it is as follows to win 50 routine patient's sample data:
| Contrast agents | Self-made reagent | |
| 1 | 75.38 | 76.78 |
| 2 | 127.91 | 124.44 |
| 3 | 28.76 | 24.31 |
| 4 | 233.24 | 235.09 |
| 5 | 139.95 | 137.59 |
| 6 | 140.1 | 138.03 |
| 7 | 93.65 | 96.81 |
| 8 | 195.83 | 190.88 |
| 9 | 48.15 | 47.61 |
| 10 | 76.1 | 82.17 |
| 11 | 187.11 | 186.87 |
| 12 | 129.19 | 127.43 |
| 13 | 87.84 | 80.55 |
| 14 | 255.68 | 263.45 |
| 15 | 83.88 | 81.24 |
| 16 | 50.56 | 48.12 |
| 17 | 286.43 | 291.3 |
| 18 | 201.17 | 200.48 |
| 19 | 120.84 | 121.88 |
| 20 | 243.08 | 246.27 |
| 21 | 17.95 | 17.05 |
| 22 | 20.82 | 18.78 |
| 23 | 62.39 | 61.25 |
| 24 | 25.74 | 24.15 |
| 25 | 39.57 | 37.46 |
| 26 | 15.7 | 14.73 |
| 27 | 89.77 | 90.51 |
| 28 | 153.81 | 154.9 |
| 29 | 14.74 | 15.48 |
| 30 | 49.31 | 49.19 |
| 31 | 74.23 | 73.27 |
| 32 | 37.96 | 36.92 |
[0104]
| 33 | 344.19 | 354.75 |
| 34 | 115.58 | 117.57 |
| 35 | 250.05 | 254.46 |
| 36 | 238.46 | 244.89 |
| 37 | 110.7 | 111.32 |
| 38 | 214.2 | 218.45 |
| 39 | 20.5 | 21.3 |
| 40 | 31 | 30.6 |
| 41 | 55.8 | 56.4 |
| 42 | 80.3 | 81.3 |
| 43 | 121.4 | 121.8 |
| 44 | 151.9 | 150.4 |
| 45 | 180.2 | 181.6 |
| 46 | 201.5 | 199.8 |
| 47 | 343.2 | 342.6 |
| 48 | 102.3 | 101.8 |
| 49 | 189.3 | 190.2 |
| 50 | 64.5 | 66.8 |
The more anti-cholerythrin of kit of the present invention and contrast agents, triglyceride effect better (Fig. 2, Fig. 3), interference free performance is compared as follows:
Anti-cholerythrin effect relatively
Anti-triglyceride relatively
Stability is (Fig. 4) relatively:
| 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
| |
100 | 99.6 | 100.3 | 98.6 | 98.1 | 96.2 | 94.8 | 94.1 |
| Relevant | 100 | 95.3 | 92.3 | 89.2 | 85.2 | 75.6 | 64.5 | 52.1 |
The successful of anti-cholerythrin effect of the present invention and anti-triglyceride is better than contrast agents, and 37 degree accelerated stabilities obviously are better than contrast agents.
Therefore, find surprisingly by introducing the combination of potassium ferrocyanide and surfactant, significantly promoted among the present invention reagent with respect to the anti-cholerythrin of contrast agents and the effect of anti-triglyceride.
The chromogen 3-hydroxyl-2,4 that uses among the present invention, 6-trihydroxybenzoic acid (HTIB) sensitivity is higher; It is more simple and convenient to remove sugar system glucose oxidase-hydrogen peroxidase; Having added the stabilizing agents such as Sodium azide makes reagent more stable; It is more desirable to have added the effect that potassium ferrocyanide and surfactant disturb the anti-cholerythrin effect of reagent and anti-triglyceride.The present invention significantly is better than contrast agents at above-mentioned aspect of performance.
With 1, (1, level 5-AG) is that 78umol/L is as critical value, when 1 to 5-dewatered grape sugar alcohol, it was highly sensitive when the level of 5-dewatered grape sugar alcohol was lower than 78umol/L and is diagnosed as diabetes, specificity good, compares the diagnosis index that is more suitable for diabetes with other experimental techniques.
Annotate: except other offers some clarification on, the number percent that the present invention mentions is the quality percent by volume.
Above embodiment is to explanation of the present invention and further explains, rather than limitation of the present invention, and any modification of making in spirit of the present invention and rights protection scope all falls into protection scope of the present invention.
Claims (8)
1. method of measuring 1,5-dehydration glucitol in blood content, described method are by following reaction assay 1,5-dehydration glucitol in blood content:
Wherein, utilize the glucose in glucose oxidase and the hydrogen peroxidase removing blood, utilize subsequently pyranose oxidase catalysis 1,5-dewatered grape sugar alcohol produces hydrogen peroxide, chromogen and hydrogen peroxide produce coloured substrate under the catalytic action of horseradish peroxidase, carry out colorimetric analysis and determine thus the content of 1,5-dehydration glucitol in blood
The combination that described method further comprises the steps: to add potassium ferrocyanide and surfactant is eliminated in the sample high cholerythrin to the impact of sample measured value.
2. mensuration blood 1 according to claim 1, the method of 5-dewatered grape sugar alcohol content, further comprise the steps: to add surfactant and eliminate in the sample triglyceride to the impact of sample measured value, and/or add ascorbic acid oxidase and eliminate in the sample high ascorbic acid to the impact of sample.
3. measure blood 1 for one kind, the kit of 5-dewatered grape sugar alcohol content, comprise reagent I and reagent II two parts, wherein the reagent I contains glucose oxidase, hydrogen peroxidase, ascorbic acid oxidase, surfactant, potassium ferrocyanide, damping fluid, antiseptic, stabilizing agent; The reagent II contains pyranose oxidase, 4-AAP, horseradish peroxidase, 3-hydroxyl-2,4,6-Triiodobenzoic acid, damping fluid, antiseptic, stabilizing agent.
4. the content of each component is in the kit of mensuration 1,5-dehydration glucitol in blood content according to claim 3, every liter of reagent I solution:
The content of each component is in every liter of reagent II:
5. the content of each component is in the kit of mensuration 1,5-dehydration glucitol in blood content according to claim 3, every liter of reagent I solution:
The content of each component is in every liter of reagent II:
6. mensuration blood 1 according to claim 1, the method of 5-dewatered grape sugar alcohol content, wherein said surfactant is selected from one or more in triton x-100, B66, polysorbas20, neopelex, betaine, the cetyl trimethyl ammonium bromide.
7. each described mensuration blood 1 according to claim 3-5, the kit of 5-dewatered grape sugar alcohol content, wherein said surfactant is selected from one or more in triton x-100, B66, polysorbas20, neopelex, betaine, the cetyl trimethyl ammonium bromide.
8. each described mensuration blood 1 according to claim 3-5, the kit of 5-dewatered grape sugar alcohol content, wherein said stabilizing agent and antiseptic are selected from one or more in Sodium azide, nitrine lithium, PC300, Sodium Benzoate, potassium sorbate, propylparaben, polyglycol, polyvinyl alcohol (PVA), the disodium ethylene diamine tetraacetate.
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| EP0261591A2 (en) * | 1986-09-22 | 1988-03-30 | Nippon Kayaku Kabushiki Kaisha | Method for assaying 1,5-anhydroglucitol and kit therefor |
| CN101071105A (en) * | 2007-06-12 | 2007-11-14 | 冯景 | Method for determining glucose and 1,5-anhydroglucitol in identicial colorimetric cell |
| CN101558296A (en) * | 2006-12-14 | 2009-10-14 | 日本化药株式会社 | Method for measuring 1,5-anhydroglucitol in whole blood, and sensor chip and measurement kit to be used in the method |
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| EP0261591A2 (en) * | 1986-09-22 | 1988-03-30 | Nippon Kayaku Kabushiki Kaisha | Method for assaying 1,5-anhydroglucitol and kit therefor |
| CN101558296A (en) * | 2006-12-14 | 2009-10-14 | 日本化药株式会社 | Method for measuring 1,5-anhydroglucitol in whole blood, and sensor chip and measurement kit to be used in the method |
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