CN105021543B - A kind of alpha-amylase detection reagent and its application - Google Patents
A kind of alpha-amylase detection reagent and its application Download PDFInfo
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Abstract
The invention belongs to technical field of medical examination, and in particular to a kind of alpha amylase detection reagent and its application.The reagent includes reagent R1 and reagent R2, and the reagent R1 is mainly by MES buffer solutions, NH4Cl, CaCl2, surfactant and preservative composition；The reagent R2 is mainly made of MES buffer solutions, 2 chlorine, 4 nitrobenzene maltotriose and preservative.Alpha amylase detection reagent provided by the invention does not have NaN3, and the allosteric of alpha amylase will not be caused in continuous mode, it is possible to reduce the error of measurement result；Simultaneously toolenzyme need not be added in detection process, delay time is short, and testing result repeatability and stablize, be conducive to the clinical application of the alpha amylase detection reagent.
Description
Technical field
The invention belongs to technical field of medical examination, and in particular to a kind of alpha-amylase detection reagent and its application.
Background technology
Alpha-amylase scientific name is Isosorbide-5-Nitrae-α-D- glucan -4- glucan hydrolases, is divided by having in salivary gland and pancreas
Secrete the enzyme of the epithelial cell synthesis of function.Alpha-amylase is to detect an important biochemical marker of acute pancreatitis.Therefore,
The concentration for measuring alpha-amylase has important clinical meaning.
According to statistics, the assay method of alpha-amylase probably has more than 200 kinds, is broadly divided into viscosimetry, iodimetric titration, than turbid
The six major class assay method such as method, mashing system, dyestuff method for releasing and fluorescence method.Constantly go deep into to alpha-amylase assay method
, there are many new assay methods in research.Currently, using method being the most ethylidene -4-NP- maltoheptaose glycosides（EPS）
Method or direct measuring method.Due to ethylidene -4-NP- maltoheptaose glycosides（EPS）Method is needed there are reagent working solution stability is poor
The shortcomings that alpha-glucosidase toolenzyme and gradually by with toolenzyme need not be added, delay time is short, stable reagent it is straight
Measuring method is connect to be replaced.
Chinese patent application 201310103140.7 discloses alpha-amylase detection reagent and the application of a kind of stabilization, described
Alpha-amylase detection reagent includes that volume ratio is 4:1 buffer solution and substrate solution, the buffer solution by MES buffer solutions, NaCl,
CaCl2, neopelex and Liquid BPF aN3 be formulated according to a certain ratio；The substrate solution by MES buffer solutions,
The chloro- 4- nitrobenzenes of 2--maltoside (Gal-G2-CNP), neopelex and Liquid BPF aN3 matches according to a certain ratio
It makes.The alpha-amylase detection reagent need not add toolenzyme using the chloro- 4- nitrobenzenes-maltosides of 2- as substrate, together
When with the non-reducing end residue of the sugar-modified Fructus Hordei Germinatus oligose glycosides of gala, prevent the non-specific hydrolysis of substrate, bottom can be significantly improved
The stability of object, to avoid can endogenous glucose glycosides enzyme influence testing result；But the NaN3 in reagent is to alpha-amylase
There is isomerization, the allosteric of alpha-amylase can be caused in continuous mode, leads to measuring there are larger error for alpha-amylase,
Measurement result is less reproducible, to influence the accuracy of acute pancreatitis diagnosis.
Therefore, research hair, which outputs one kind, need not be added toolenzyme, and delay time is short, stable reagent, measurement result error
Small, reproducible alpha-amylase detection reagent is the problem of current urgent need to resolve.
Invention content
In order to overcome prior art alpha-amylase detection reagent there are measurement result error is larger, measurement result poor repeatability
The shortcomings that, the object of the present invention is to provide a kind of alpha-amylase detection reagent and its applications, to solve the above problem.
A kind of alpha-amylase detection reagent provided by the invention, the reagent include reagent R1 and reagent R2, the reagent R1
Including following components：
MES buffer solutions 40-100mmol/L, NH4Cl 80-160mmol/L、CaCl25-30mmol/L, surfactant
2-12g/L and preservative 2-8g/L；
The reagent R2 includes following components：
The chloro- 4- nitrobenzenes-maltotriose 10-80mmol/L and preservative 2-8g/ of MES buffer solutions 40-100mmol/L, 2-
L。
Preferably, alpha-amylase detection reagent provided by the invention includes reagent R1 and reagent R2, the reagent R1 include
Following components：
MES buffer solutions 80mmol/L, NH4Cl 120mmol/L、CaCl210mmol/L, surfactant 5g/L and anti-corrosion
Agent 6g/L；
The reagent R2 includes following components：
The chloro- 4- nitrobenzenes-maltotriose 30mmol/L and preservative 5g/L of MES buffer solutions 80mmol/L, 2-.
Further, the pH value of MES buffer solutions provided by the invention is 5.5-6.5, and the pH value of MES buffer solutions is preferably
6.0。
Further, surfactant provided by the invention is polyethylene glycol.Polyethylene glycol is a kind of nontoxic, non-stimulated
Property, there is good water-soluble surfactant, the stability of reagent can be increased.
Further, preservative provided by the invention is p-hydroxybenzoate.Since p-hydroxybenzoate has phenol
Hydroxyl structure has significant antibacterial action, can effectively extend shelf life of products.
MES buffer solutions provided by the invention are 2-morpholine ethane sulfonic acid（MES）The molecular formula of buffer solution, 2-morpholine ethane sulfonic acid is
C6H13NO4S, No. CAS is 4432-31-9.
Alpha-amylase detection reagent provided by the invention does not contain sodium azide, and alphalise starch will not be caused in continuous mode
The allosteric of enzyme, it is possible to reduce the error of measurement result；And alpha-amylase detection reagent provided by the invention has stable reagent,
Toolenzyme and delay time short advantage need not be added, is a kind of easy to operate, the accurate alpha-amylase detection of measurement result
Reagent is conducive to clinical application.
Tests prove that alpha-amylase detection reagent provided by the invention has measurement result reproducible, stability
Good advantage is a kind of ideal detection reagent for measuring alpha-amylase concentration.
In addition, the present invention also provides a kind of detection method of alpha-amylase detection reagent, include the following steps：It will prepare
Good reagent R1 and reagent R2 is placed on the corresponding reagent position of automatic clinical chemistry analyzer, is placed in the corresponding position of sample disc
Blank control product, standard items and sample obtain the absorbance change of standard items detection by reference substance and standard items, further according to complete
The absorbance for the sample that automatic biochemistry analyzer measures, calculates the alpha-amylase concentration of sample.
Further, the temperature of the automatic clinical chemistry analyzer is 35-40 DEG C.
Further, the delay time of the automatic clinical chemistry analyzer is 30-60S, reading duration 120-150S.
Further, the Detection wavelength of the automatic clinical chemistry analyzer is respectively dominant wavelength 405nm, commplementary wave length
505nm。
The testing principle of alpha-amylase detection reagent provided by the invention is：Using the chloro- 4- nitrobenzenes-maltotrioses of 2- as
The substrate of alpha-amylase, the maltose terminal molecular of the chloro- 4- nitrobenzenes-maltotrioses of α-amylasehydrolysis 2-, directly generates one
Kind yellow uitramarine indicator, the increase that its absorbance is measured at 405nm are directly proportional to alpha-amylase concentration in sample.
In short, alpha-amylase detection reagent provided by the invention, has following advantage compared with prior art：
1）Alpha-amylase detection reagent provided by the invention does not contain sodium azide, and α-shallow lake will not be caused in continuous mode
The allosteric of powder enzyme, it is possible to reduce the error of measurement result；
2）Alpha-amylase detection reagent provided by the invention has stable reagent, need not add toolenzyme and delay time
Short advantage；
3）Alpha-amylase detection reagent testing result provided by the invention is reproducible, and stability is good, is a kind of ideal
The detection reagent for measuring alpha-amylase concentration, is conducive to the clinical application of the alpha-amylase detection reagent.
Description of the drawings
Fig. 1, which is that embodiment 2- reagents are linearly related with GmbH companies-reagent, to scheme.
Specific implementation mode
The present invention is further described below by way of specific embodiment, the present invention is not limited only to following embodiment.In this hair
In bright range or present disclosure is not being departed from, in spirit and scope, the change that is carried out to the present invention is combined or replaced
It changes, will be apparent to the person skilled in the art, and be included within the scope of the present invention.
Embodiment 1, alpha-amylase detection reagent
Alpha-amylase detection reagent includes reagent R1 and reagent R2, and the reagent R1 includes following components：
MES buffer solutions 40mmol/L, NH that pH value is 5.54Cl 80mmol/L、CaCl25mmol/L, polyethylene glycol 3g/
L, p-hydroxybenzoate 2g/L；
The reagent R2 includes following components：
PH value be 5.5 the chloro- 4- nitrobenzenes-maltotriose 10mmol/L of MES buffer solutions 40mmol/L, 2-, to hydroxyl
Benzoic ether 2g/L.
Embodiment 2, alpha-amylase detection reagent
Alpha-amylase detection reagent includes reagent R1 and reagent R2, and the reagent R1 includes following components：
MES buffer solutions 80mmol/L, NH that pH value is 6.04Cl 120mmol/L、CaCl210mmol/L, polyethylene glycol
5g/L, p-hydroxybenzoate 6g/L；
The reagent R2 includes following components：
PH value be 6.0 the chloro- 4- nitrobenzenes-maltotriose 30mmol/L of MES buffer solutions 80mmol/L, 2-, to hydroxyl
Benzoic ether 5g/L.
Embodiment 3, alpha-amylase detection reagent
Alpha-amylase detection reagent includes reagent R1 and reagent R2, and the reagent R1 includes following components：
MES buffer solutions 100mmol/L, NH that pH value is 6.54Cl160mmol/L、CaCl230mmol/L, polyethylene glycol
10g/L, p-hydroxybenzoate 8g/L；
The reagent R2 includes following components：
PH value be 6.5 the chloro- 4- nitrobenzenes-maltotriose 80mmol/L of MES buffer solutions 100mmol/L, 2-, to hydroxyl
Benzoic ether 8g/L.
The correlation test of test example one, alpha-amylase detection reagent
1, test reagent：Alpha-amylase detection reagent prepared by embodiment 2, what State Food and Drug Administration was approved
α-determination of amylase reagent of DiaSysDiagnostic Systems GmbH companies.
2, test method：The alpha-amylase detection reagent prepared using embodiment 2, it is total with state food pharmaceuticals administration
The alpha-amylase assay kit (EPS-G7 methods) for the DiaSysDiagnostic Systems GmbH companies that office is approved, is adopted
It measures 20 clinical serum samples simultaneously with 7170 automatic biochemistry analyzer of Hitachi and carries out control test, obtain the correlation of two kinds of reagents
Linearity curve.The parameter that vertical 7170 automatic biochemistry analyzers are set as：Temperature is 37 DEG C, and delay 60s starts read point, and the reading duration is about
120s, Detection wavelength are respectively dominant wavelength 405nm, commplementary wave length 505nm.
3, test result：
For test result as shown in Figure 1, result calculates after measured, the related coefficient of two kits is 0.9997, is illustrated
The two has great correlation, to prove that alpha-amylase detection reagent provided by the invention has good practicability.
The stability test of test example two, alpha-amylase detection reagent
1, test reagent：Alpha-amylase detection reagent prepared by embodiment 2, standard items：Switzerland Roche cfas calibrates serum,
Quality-control product：The high and low quality-control product provided by Bio-Rad companies of the U.S..
2, test method：Using 7170 automatic biochemistry analyzer of Hitachi measure, the parameter set as：Temperature is 37 DEG C,
Delay 60s starts read point, reading duration about 120s, and Detection wavelength is respectively dominant wavelength 405nm, commplementary wave length 505nm.First use standard
Product are calibrated, and the alpha-amylase detection reagent for reusing the preparation of embodiment 2 measures high and low two horizontal quality-control products, lasting to measure half
Year, according to determination data, calculate test mean value and the coefficient of variation.
3, test result
Test result is as shown in table 1.
The test result data of 1 high and low quality-control product of table
| Mean value（U/L） | The coefficient of variation（%） | |
| High level quality-control product | 290.2U/L | 4.89% |
| Low value quality-control product | 30.9U/L | 3.54% |
It is obtained by table 1, the coefficient of variation of the high and low quality-control product of alpha-amylase detection reagent detection provided by the invention is less than
5%, it was demonstrated that the measurement result of alpha-amylase detection reagent of the invention is with good stability.
The repetitive test of test example three, alpha-amylase detection reagent
1, test reagent：Alpha-amylase detection reagent prepared by embodiment 2, standard items：Switzerland's Roche just occurs to calibrate blood
Clearly, sample：Collect the serum of clinical high and low level.
2, test method：Using 7170 automatic biochemistry analyzer of Hitachi measure, the parameter set as：Temperature is 37 DEG C,
Delay 60s starts read point, reading duration about 120s, and Detection wavelength is respectively dominant wavelength 405nm, commplementary wave length 505nm.First use standard
Product are calibrated, and the alpha-amylase detection reagent for reusing the preparation of embodiment 2 measures the serum 20 times of the clinical high and low level collected,
It carries out batch interior repetitive test and test mean value and the coefficient of variation is calculated according to detection data.
3, test result
Test result is as shown in table 2.
The serum test result data of 2 high and low level of table
| Mean value（U/L） | The coefficient of variation（%） | |
| High level serum | 133.4U/L | 0.72% |
| Low value serum | 24.6U/L | 1.52% |
It is obtained by table 2, the coefficient of variation of the high and low value serum of alpha-amylase detection reagent detection provided by the invention is less than
5%, it was demonstrated that the measurement result of alpha-amylase detection reagent of the invention has good repeatability.
Claims (8)
1. a kind of alpha-amylase detection reagent, which is characterized in that the reagent include reagent R1 and reagent R2, the reagent R1 by with
The following group is grouped as：
MES buffer solutions 40-100mmol/L, NH4Cl 80-160mmol/L、CaCl2 5-30mmol/L, surfactant 2-
12g/L and preservative 2-8g/L, the surfactant are polyethylene glycol, and the preservative is p-hydroxybenzoate；
The reagent R2 is composed of the following components：
The chloro- 4- nitrobenzenes-maltotriose 10-80mmol/L and preservative 2-8g/L of MES buffer solutions 40-100mmol/L, 2-,
The preservative is p-hydroxybenzoate.
2. alpha-amylase detection reagent as described in claim 1, which is characterized in that the reagent includes reagent R1 and reagent R2,
The reagent R1 is composed of the following components：
MES buffer solutions 80mmol/L, NH4Cl 120mmol/L、CaCl2 10mmol/L, surfactant 5g/L and preservative
6g/L；
The reagent R2 is composed of the following components：
The chloro- 4- nitrobenzenes-maltotriose 30mmol/L and preservative 5g/L of MES buffer solutions 80mmol/L, 2-.
3. alpha-amylase detection reagent as claimed in claim 1 or 2, which is characterized in that the pH value of the MES buffer solutions is
5.5-6.5。
4. alpha-amylase detection reagent as claimed in claim 3, which is characterized in that the pH value of the MES buffer solutions is 6.0.
5. a kind of detection method of alpha-amylase detection reagent as described in claim 1-4 is any, which is characterized in that including with
Lower step：
Prepared reagent R1 and reagent R2 are placed on the corresponding reagent position of automatic clinical chemistry analyzer, in pair of sample disc
Position is answered to place blank control product, standard items and sample, the absorbance that standard items detection is obtained by reference substance and standard items becomes
Change, further according to the absorbance for the sample that automatic clinical chemistry analyzer measures, calculates the alpha-amylase concentration of sample.
6. the detection method of alpha-amylase detection reagent as claimed in claim 5, which is characterized in that the full-automatic biochemical point
The temperature of analyzer is 35-40 DEG C.
7. the detection method of alpha-amylase detection reagent as claimed in claim 5, which is characterized in that the full-automatic biochemical point
The delay time of analyzer is 30-60s, reading duration 120-150s.
8. the detection method of as claimed in claim 5-Amylase detection reagent, which is characterized in that the full-automatic biochemical point
The Detection wavelength of analyzer is respectively dominant wavelength 405nm, commplementary wave length 505nm.
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| CN108982885B (en) * | 2017-06-05 | 2021-11-16 | 广州东林生物科技有限公司 | Kit for measuring alpha-amylase in serum |
| CN109001189B (en) * | 2017-06-06 | 2021-04-27 | 中粮营养健康研究院有限公司 | Method for detecting amylase content range in sugar product prepared from sugarcane and application of method |
| CN107828855B (en) * | 2017-10-31 | 2020-08-21 | 南京欣迪生物药业工程有限责任公司 | Seminal plasma neutral alpha-glucosidase detection kit and application thereof |
| CN110951824B (en) * | 2019-11-15 | 2023-08-11 | 中山市创艺生化工程有限公司 | Application of composite buffer system in preparation of alpha-amylase assay kit |
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| WO2009100107A2 (en) * | 2008-02-05 | 2009-08-13 | Actavis Group Ptc Ehf | Alendronate formulations, method of making and method of use thereof |
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| CN104198691A (en) * | 2014-08-14 | 2014-12-10 | 上海睿康生物科技有限公司 | Stable alpha-amylase detection kit |
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