CN102288559A - Method and kit for detecting amylase - Google Patents
Method and kit for detecting amylase Download PDFInfo
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- CN102288559A CN102288559A CN2011101534178A CN201110153417A CN102288559A CN 102288559 A CN102288559 A CN 102288559A CN 2011101534178 A CN2011101534178 A CN 2011101534178A CN 201110153417 A CN201110153417 A CN 201110153417A CN 102288559 A CN102288559 A CN 102288559A
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Abstract
The invention relates to the technical field of medical detection and discloses a method and a kit for detecting amylase. The method for detecting the amylase provided by the invention comprises the following steps of: in a buffer solution with a pH value of 7.2, mixing a sample to be detected and soluble starch to obtain a mixed solution; mixing potassium iodide and acidic potassium iodate and mixing the mixture of the potassium iodide and the acidic potassium iodate with the obtained mixed solution; detecting absorbance in a 630nm wavelength; and calculating the concentration of the amylase in the sample to be detected according to the absorbance of a non-enzymatic blank tube which is subjected to the same treatment. In the method, the potassium iodide and the potassium iodate generate iodine under the acidic condition, and the iodine is combined with the unreacted starch to form a blue compound, so influence on reaction color development due to reduction of components caused by easiness in sublimation and volatilization of an iodine solution which is directly used is avoided, and the accuracy of a detection result is improved. The kit is good in stability, long in preservation time, wide in application range and convenient for popularization and can be applied to determination of the content of the amylase in serum or urine by hospitals, health departments and medical biological scientific research institutes at all levels.
Description
Technical field
The present invention relates to medical test determination techniques field, relate to diastatic method of a kind of detection and kit specifically.
Background technology
Diastase (α-Amylase) is called for short AMS or AMY, and a class can be with the enzyme of starch molecule hydrolysis, and general action is in α-1 such as soluble starch, amylose, glycogen, the 4-glucosan, and hydrolyzing alpha-1, the 4-glycosidic bond produces glucosan, maltose and glucose molecule.Diastase content in pancreas and salivary gland is abundant, and amylopsin enters alimentary canal by pancreas with activated state, is the enzyme of most important hydrolysis carbohydrates, with the same AMS that all belongs to of diastase of salivary gland secretion.The diastase molecular weight is little, can pass through glomerular filtration, discharges with urine.Can cause that when suffering from pancreatic disease or the exocrine pancreatic function obstacle is arranged amylase activity raises or reduction, therefore, the diastase of measuring in serum and the urine is significant to pancreatitic diagnosis.The fluctuation of amylase in urine level is bigger, thus preferred serum amylase detection, or both measure simultaneously.
The assay method of amylase activity has a variety ofly in serum and the urine, mainly contains viscosimetry, turbidimetry, mashing system, iodine-starch colourimetry, enzyme rate method and dyestuff method for releasing etc.Wherein, viscosimetry and turbidimetry lack specificity and sensitivity because influence factor is more, are eliminated.Though mashing system can directly be measured enzymatic activity, complicated operation is not suitable for routine clinical inspection.At present widely used is iodimetric titration, enzyme rate method and dyestuff method for releasing.Wherein, cost is low because of having, detection does not need expensive instrument, stable reagent, easy and simple to handle, reliable results for the iodine-starch colourimetry, is used widely clinically.
Diastatic reaction principle is α-1,4 glycosidic bond hydrolysis in the AMS catalysis starch molecule in serum (slurry) and the urine in iodine-starch colorimetric method for determining serum and the urine, produces glucose, maltose and contains the dextrin of α-1,6 glycosidic bond side chain.Under the quantitative condition of substrate, the reaction back adds iodine liquid and is combined into blue compound with the starch that is not hydrolyzed, its blue depth with without the blank pipe of enzymatic reaction relatively, amylase activity is big more, remaining starch is few more, and reactant liquor colour developing is shallow more, places final product under the ultraviolet analyser or under the semi-automatic biochemical analyzer, detection is in the absorbance size at predominant wavelength 630nm, thus diastatic content in the measuring and calculating sample.
Volatilization causes composition to reduce the thin out reaction solution that influences yet said method developer iodine liquid easily distils, and causes the testing result accuracy to reduce.
Summary of the invention
In view of this, the object of the invention provides the kit of the detection diastase enzyme that a kind of stability is high, accuracy is high.
For realizing purpose of the present invention, the present invention adopts following technical scheme:
The diastatic method of a kind of detection comprises:
Step 1, in the damping fluid of pH7.2, testing sample mixes with soluble starch and obtains mixed liquor;
Step 2, potassium iodide mix with acid Potassiumiodate, and the mixed liquor reaction that obtains with step 1 detects absorbance under the 630nm wavelength, according to the absorbance without the blank pipe of enzymatic through above-mentioned same treatment, calculate diastatic concentration in the testing sample.
α-1,4 glycosidic bond hydrolysis in the AMS catalysis starch molecule in serum (slurry) and the urine produces glucose, maltose and contains the dextrin of α-1,6 glycosidic bond side chain.Under the quantitative condition of substrate, the reaction back adds iodine liquid and is combined into blue compound with the starch that is not hydrolyzed, its blue depth with without the blank pipe of enzymatic reaction relatively, detect and be in the absorbance size at predominant wavelength 630nm, thus diastatic content in the measuring and calculating sample.Detection method of the present invention with potassium iodide and acid potassium iodide as colour developing liquid, potassium iodide and Potassiumiodate generate iodine under acid condition, combine the blue compound of formation with unreacted starch, avoid directly using the volatilization that easily distils of iodine liquid to cause composition to reduce the thin out reaction solution that influences, improve the testing result accuracy.
Preferably, the mol ratio of described Potassiumiodate and potassium iodide is 1: 5～6.
Preferably, acid Potassiumiodate is the potassium iodate solution of 8～12mmol/LpH2.0～3.0.
For the good combination of enzyme-to-substrate, enzyme and substrate all need damping fluid that the suitable environment that dissociates is provided.The ionic strength of damping fluid also affects the activity of enzyme, and ionic strength is too high, electrolyte interferases and substrate combination, enzymatic activity will progressively descend, but ionic strength cross low also possibly can't the kinase activity.The more approaching ionic strength of body fluid of general selection and physiological environment.Therefore detection method of the present invention is in order to provide a suitable enzymolysis environment, and testing sample mixes in the damping fluid of pH7.2 with soluble starch.Described damping fluid at pH7.2 comprises: sodium hydrogen phosphate-citrate buffer solution, Tris-hydrochloride buffer and phosphate buffer.As preferably, the buffer solution of pH7.2 is the phosphate buffer of 50～100mmol/L pH7.2 described in the detection method of the present invention, comprises sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution and sodium hydrogen phosphate-potassium phosphate buffer.
Preferably, described soluble starch concentration is 960mg/L.
The present invention also provides a kind of detection diastatic kit, comprises the phosphate buffer of 960mg/L soluble starch, 50～100mmol/L pH7.2, potassium iodate solution and 30～50mmol/L potassium iodide of 8～10mmol/LpH2.0～3.0.
Preferably, kit of the present invention also comprises the Sodium Benzoate of 50～70mmol/L, and Sodium Benzoate is as antiseptic guarantee reagent stability.
The diastatic kit of detection provided by the invention can be double reagent, phosphate buffer and the potassium iodide of soluble starch and pH7.2 are made first reagent, acid Potassiumiodate is made second reagent, when utilizing this kit to detect diastase, with first reagent and second reagent mix, thus the diastase in the detection testing sample.The diastatic kit of detection provided by the invention can also be many reagent, the phosphate buffer of soluble starch and pH7.2 is made first reagent, acid Potassiumiodate is made second reagent, potassium iodide is made the 3rd reagent, when detecting diastase, with first reagent and second reagent and the 3rd reagent mix, detect the diastase in the testing sample.Because the soluble starch aqueous stability is poor, therefore kit of the present invention is made powdered reagent with the reagent of soluble-containing starch, dissolving before using.
From above-mentioned technical scheme as can be seen, detection method of the present invention with potassium iodide and acid potassium iodide as colour developing liquid, potassium iodide and Potassiumiodate generate iodine under acid condition, combine the blue compound of formation with unreacted starch, avoid directly using the volatilization that easily distils of iodine liquid to cause composition to reduce the thin out reaction solution that influences, improve the testing result accuracy.Test shows, detection method of the present invention has high accuracy and precision, same sample is carried out repeatability to be detected, standard rate (coefficient of variation) between each result is 1.92%, less than 3% of standard, and the range of linearity is wide, and diastase survey line scope can reach 800U/L in the serum, and diastase survey line scope can reach 1600U/L in the urine.Kit good stability of the present invention, long shelf-life, applied widely, be convenient to promote the use of, can be applicable to hospitals at different levels, sanitary precaution department and medical biotechnology R﹠D institution and measure diastase content in serum or the urine.
Embodiment
The embodiment of the invention discloses diastatic method of a kind of detection and kit.Those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Product of the present invention and method are described by preferred embodiment, and the related personnel obviously can change or suitably change and combination method as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
In order further to understand the present invention, the present invention is described in detail below in conjunction with embodiment.
Embodiment 1: the diastatic kit of detection of the present invention.
The diastatic kit of detection of the present invention is many reagent.
First reagent is powdered reagent, comprises phosphate buffer and the 60mmol/L Sodium Benzoate of 960mg/L soluble starch, 50mmol/LpH7.2;
Second reagent is the 30mmol/L potassium iodide;
The 3rd reagent is the potassium iodate solution of 8mmol/LpH2.0.
Embodiment 2: the diastatic kit of detection of the present invention.
The diastatic kit of detection of the present invention is many reagent.
First reagent is powdered reagent, comprises phosphate buffer and the 50mmol/L Sodium Benzoate of 960mg/L soluble starch, 20mmol/LpH7.2;
Second reagent is the 50mmol/L potassium iodide;
The 3rd reagent is the potassium iodate solution of 12mmol/LpH3.0.
Embodiment 3: the diastatic kit of detection of the present invention.
The diastatic kit of detection of the present invention is a double reagent.
First reagent is powdered reagent, comprises phosphate buffer, 48mmol/L potassium iodide and the 70mmol/L Sodium Benzoate of 960mg/L soluble starch, 100mmol/LpH7.2;
Second reagent is the potassium iodate solution of 8mmol/LpH2.5.
Embodiment 4: detect diastatic method.
Set 37 ℃ of semi-automatic biochemical analyzer temperature of reaction, reaction method is an end-point method, measures predominant wavelength 630nm, and the Direction of Reaction is negative reaction.Getting testing sample mixes with the 960mg/L soluble starch, hatch 360s, add the acid Potassiumiodate mixed solution of 40mmol/L potassium iodide and 8mmol/LpH2.0 then, mix and be placed on semi-automatic biochemical analyzer, detect and write down the absorbance under the 630nm wavelength.Blank pipe without enzymatic is identical with above-mentioned processing.According to computing formula C
Sample=(A
Blank-A
Sample)/A
Blank* (C * V)/(t * V
Sample) calculate diastatic concentration in the testing sample, wherein, C
SampleBe sample to be tested concentration; A
SampleBe the sample absorbance; A
BlankBe blank pipe absorbance; C is a soluble starch concentration; V is the soluble starch volume; T is reaction time (min); V
SampleBe the testing sample volume.Because every liter of example reaction 1min hydrolysis 4mg starch of international unit regulation is 1 international unit (U/L), therefore in order to be that international unit need be according to formula C with gained diastase concentration conversion
Sample (international unit)=C
Sample* (1 * 1000)/4, wherein 1 reaction time (min) for the international unit regulation; 1000 are converted into the factor of L for mL; 4 every liter of sample hydrolysis amount of starch for the international unit regulation.
Embodiment 5: detection method analysis of the accuracy of the present invention
Get 20 routine clinical samples, respectively according to embodiment 4 described method and Gal-G
2-CNP substrate method is utilized the diastase content in the CB171 semi-automatic biochemical analyzer test sample, the results are shown in Table 1.
Table 1 sample diastase content detection result
By table 1 result as seen, with existing Gal-G
2-CNP substrate method is compared, and detection method testing result of the present invention is close, and related coefficient is 0.995, and detection method testing result of the present invention accurately and reliably.
Embodiment 6: detection method repeatability of the present invention detects
As testing sample,, utilize the diastase content in the CB171 semi-automatic biochemical analyzer detection testing sample, with conventional serum sample with existing Gal-G according to embodiment 4 described methods
2-CNP substrate method method relatively the results are shown in Table 2.
Table 2 sample repeatability testing result
| Detection method of the present invention | Gal-G 2-CNP substrate method |
| 124 | 127 |
| 121 | 121 |
| 122 | 124 |
| 118 | 119 |
| 124 | 124 |
| 120 | 122 |
| 121 | 121 |
| 117 | 120 |
| 119 | 125 |
| 121 | 122 |
| CV％＝1.92％ | CV％＝2.01％ |
By the result of table 2 as seen, adopt detection method of the present invention to detect diastatic coefficient of variation CV=1.92%,, meet " the general requirement of external diagnosis reagent " less than 5%, and less than existing Gal-G
2-CNP substrate method the coefficient of variation shows detection method good reproducibility of the present invention.
Embodiment 7: the detection of the method for the invention linearity
Get the high value serum of diastase concentration near 800U/L, be diluted to 5 different concentration gradients, the theoretical concentration value is followed successively by 50,100,200,400,800U/L, according to embodiment 4 described methods, utilize the diastase in the CB171 semi-automatic biochemical analyzer detection testing sample, each concentration determination 2 times is averaged, calculate the correlation coefficient r value according to formula, the results are shown in Table 3.
Get the high value urine of diastase concentration near 1600U/L, be diluted to 5 different concentration gradients, the theoretical concentration value is followed successively by 100,200,400,800,1600U/L, according to embodiment 4 described methods, utilize the diastase in the CB171 semi-automatic biochemical analyzer detection testing sample, each concentration determination 2 times is averaged, calculate the correlation coefficient r value according to formula, the results are shown in Table 4.
Table 3 blood serum sample result of linear detection
Table 4 urine sample result of linear detection
By the result of table 3 and table 4 as seen, the diastatic range of linearity can reach 800U/L in the detection method serum of the present invention, and the diastatic range of linearity can reach 1600U/L in the urine, shows that the detection method range of linearity of the present invention is wide, applied widely.
The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Claims (7)
1. one kind is detected diastatic method, it is characterized in that, comprising:
Step 1, in the damping fluid of pH7.2, testing sample mixes with soluble starch and obtains mixed liquor;
Step 2, potassium iodide mix with acid Potassiumiodate, and the mixed liquor reaction that obtains with step 1 detects absorbance under the 630nm wavelength, according to the absorbance without the blank pipe of enzymatic through above-mentioned same treatment, calculate diastatic concentration in the testing sample.
2. according to the described detection method of claim 1, it is characterized in that the mol ratio of described Potassiumiodate and potassium iodide is 1: 4～6.
3. according to the described detection method of claim 1, it is characterized in that described acid Potassiumiodate is the potassium iodate solution of 8～12mmol/LpH2.0～3.0.
4. according to the described detection method of claim 1, it is characterized in that the buffer solution of described pH7.2 is the phosphate buffer of 50～100mmol/L pH7.2.
5. according to the described detection method of claim 1, it is characterized in that described soluble starch concentration is 960mg/L.
6. one kind is detected diastatic kit, it is characterized in that, comprises the phosphate buffer of 960mg/L soluble starch, 50～100mmol/L pH7.2, Potassiumiodate and 30～50mmol/L potassium iodide of 8～12mmol/LpH2.0～3.0.
7. according to the described kit of claim 6, it is characterized in that, also comprise the Sodium Benzoate of 50～70mmol/L.
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Cited By (8)
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| CN103472015A (en) * | 2013-09-17 | 2013-12-25 | 四川农业大学 | Dynamic absorbance quantitative analysis method based on iodine-starch chromogenic system |
| CN103789400B (en) * | 2014-02-28 | 2015-12-02 | 多多药业有限公司 | A kind of detection method of beta-amylase |
| WO2017208249A1 (en) | 2016-05-31 | 2017-12-07 | Indian Institute Of Technology, Guwahati | A transmittance based system/kit for point-of-care quantification of biomarkers sample and use thereof |
| CN107978220A (en) * | 2017-11-07 | 2018-05-01 | 阜阳师范学院 | A kind of intelligence egg shelf-life indicating label |
| CN109001189A (en) * | 2017-06-06 | 2018-12-14 | 中粮营养健康研究院有限公司 | Detect the method and its application of the amylase content range in the sugar products of sugarcane |
| CN109406512A (en) * | 2018-12-25 | 2019-03-01 | 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） | It is a kind of using xanthan gum as the detection method of amylase in the production technology of raw material |
| CN109459431A (en) * | 2018-12-25 | 2019-03-12 | 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） | The detection method of amylase in a kind of oyster sauce |
| CN111157465A (en) * | 2019-12-30 | 2020-05-15 | 武汉艾迪康医学检验所有限公司 | α -amylase detection method |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103472015A (en) * | 2013-09-17 | 2013-12-25 | 四川农业大学 | Dynamic absorbance quantitative analysis method based on iodine-starch chromogenic system |
| CN103472015B (en) * | 2013-09-17 | 2016-02-17 | 四川农业大学 | Based on iodine-starch color development system dynamic absorbance quantitative analysis method |
| CN103789400B (en) * | 2014-02-28 | 2015-12-02 | 多多药业有限公司 | A kind of detection method of beta-amylase |
| WO2017208249A1 (en) | 2016-05-31 | 2017-12-07 | Indian Institute Of Technology, Guwahati | A transmittance based system/kit for point-of-care quantification of biomarkers sample and use thereof |
| US10900909B2 (en) | 2016-05-31 | 2021-01-26 | Indian Institute Of Technology, Guwahati | Transmittance based system/kit for point-of-care quantification of biomarkers sample and use thereof |
| CN109001189A (en) * | 2017-06-06 | 2018-12-14 | 中粮营养健康研究院有限公司 | Detect the method and its application of the amylase content range in the sugar products of sugarcane |
| CN107978220A (en) * | 2017-11-07 | 2018-05-01 | 阜阳师范学院 | A kind of intelligence egg shelf-life indicating label |
| CN109406512A (en) * | 2018-12-25 | 2019-03-01 | 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） | It is a kind of using xanthan gum as the detection method of amylase in the production technology of raw material |
| CN109459431A (en) * | 2018-12-25 | 2019-03-12 | 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） | The detection method of amylase in a kind of oyster sauce |
| CN109406512B (en) * | 2018-12-25 | 2021-06-25 | 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） | Method for detecting amylase in production process by taking xanthan gum as raw material |
| CN111157465A (en) * | 2019-12-30 | 2020-05-15 | 武汉艾迪康医学检验所有限公司 | α -amylase detection method |
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