CN105021543A - Alpha-amylase detection reagent and application thereof - Google Patents
Alpha-amylase detection reagent and application thereof Download PDFInfo
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- CN105021543A CN105021543A CN201510385800.4A CN201510385800A CN105021543A CN 105021543 A CN105021543 A CN 105021543A CN 201510385800 A CN201510385800 A CN 201510385800A CN 105021543 A CN105021543 A CN 105021543A
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Abstract
The present invention belongs to the field of medical laboratory technology, and in particular relates to an alpha-amylase detection reagent and application thereof. The reagent comprises a reagent R1 and a reagent R2; the reagent R1 is mainly composed of an MES buffer, NH4Cl, CaCl2, a surfactant and a preservative; the R2 reagent consists of an MES buffer, a 2-chloro-4-nitrobenzene-maltotriose and a preservative. The alpha-amylase detection reagent provided by the invention does not contain NaN3, does not lead to alpha-amylase allosterism in the determination process, and can reduce the error in measurement results; at the same time, the test does not need additional tool enzyme, and has short delay time; the test results have good repeatability and stability. Therefore, the invention is conducive to the clinical application of alpha-amylase detection reagent.
Description
Technical field
The invention belongs to technical field of medical examination, be specifically related to a kind of AMS and detect reagent and application thereof.
Background technology
AMS formal name used at school is Isosorbide-5-Nitrae-α-D-glucosan-4-glucan hydrolase, is the enzyme synthesized by the epithelial cell in salivary gland and pancreas with secreting function.AMS detects the important biochemical marker of of acute pancreatitis.Therefore, the concentration measuring AMS has important clinical meaning.
According to statistics, the assay method of AMS probably has kind more than 200, is mainly divided into the six large class assay methods such as viscosimetry, iodimetric titration, turbidimetry, mashing system, dyestuff method for releasing and fluorescence method.Constantly further investigate along with to AMS assay method, occurred many new assay methods.At present, what apply method the most is ethylidene-4-NP-maltoheptaose glycosides (EPS) method or direct measuring method.Because ethylidene-4-NP-maltoheptaose glycosides (EPS) method exists reagent working fluid less stable, need the shortcoming of alpha-glucosidase toolenzyme and had gradually do not need add toolenzyme, time delay is short, and the direct measuring method of stable reagent replaced.
Chinese patent application 201310103140.7 discloses a kind of stable AMS and detects reagent and application, and described AMS detects reagent and comprises the damping fluid and substrate solution that volume ratio is 4:1, and described damping fluid is by MES damping fluid, NaCl, CaCl
2, neopelex and Liquid BPF aN3 formulated according to a certain ratio; Described substrate solution is formulated according to a certain ratio by MES damping fluid, 2-chloro-4-nitrobenzene-maltoside (Gal-G2-CNP), neopelex and Liquid BPF aN3.This AMS detects reagent and uses the chloro-4-nitrobenzene-maltoside of 2-not need to add toolenzyme as substrate, modify the non-reducing end residue of Fructus Hordei Germinatus oligose glycosides with galactose simultaneously, stop the non-specific hydrolysis of substrate, the stability of substrate can be significantly improved, thus avoid to affect testing result by endogenous glucose glycosides enzyme; But the NaN3 in reagent has isomerization to AMS, can cause the allosteric of AMS in mensuration process, cause the mensuration of AMS to there is larger error, measurement result repeatability is poor, thus affects the accuracy of acute pancreatitis diagnosis.
Therefore, one is outputed in research not to be needed to add toolenzyme, and time delay is short, stable reagent, and measurement result error is little, and it is the difficult problem needing solution at present badly that reproducible AMS detects reagent.
Summary of the invention
There is measurement result error comparatively greatly, the shortcoming of measurement result poor repeatability to overcome prior art AMS detection reagent, the object of this invention is to provide a kind of AMS and detecting reagent and application thereof, to solve the above problems.
A kind of AMS provided by the invention detects reagent, and this reagent comprises reagent R1 and reagent R2, and described reagent R1 comprises following component:
MES damping fluid 40-100mmol/L, NH
4cl 80-160mmol/L, CaCl
25-30mmol/L, surfactant 2-12g/L and antiseptic 2-8g/L;
Described reagent R2 comprises following component:
The chloro-4-nitrobenzene-maltotriose 10-80mmol/L and antiseptic 2-8g/L of MES damping fluid 40-100mmol/L, 2-.
Preferably, AMS provided by the invention detects reagent and comprises reagent R1 and reagent R2, and described reagent R1 comprises following component:
MES damping fluid 80mmol/L, NH
4cl 120mmol/L, CaCl
210mmol/L, surfactant 5g/L and antiseptic 6g/L;
Described reagent R2 comprises following component:
The chloro-4-nitrobenzene-maltotriose 30mmol/L and antiseptic 5g/L of MES damping fluid 80mmol/L, 2-.
Further, the pH value of MES damping fluid provided by the invention is that the pH value of 5.5-6.5, MES damping fluid is preferably 6.0.
Further, surfactant provided by the invention is polyglycol.Polyglycol is a kind of nontoxic, nonirritant, has good water miscible surfactant, can increase the stability of reagent.
Further, antiseptic provided by the invention is p-hydroxybenzoate.Because p-hydroxybenzoate has phenolic hydroxyl structure, there is significant antibacterial action, effectively can extend shelf life of products.
MES damping fluid provided by the invention is MES (MES) damping fluid, and the molecular formula of MES is C
6h
13nO
4s, No. CAS is 4432-31-9.
AMS provided by the invention detects reagent not containing sodium azide, can not cause the allosteric of AMS, can reduce the error of measurement result in mensuration process; And AMS provided by the invention detects reagent and has stable reagent, do not need to add toolenzyme and time delay short advantage, be a kind of simple to operate, measurement result accurately AMS detects reagent, is conducive to clinical application.
Prove through test, AMS provided by the invention detect reagent to have measurement result reproducible, the advantage of good stability is a kind of detection reagent of desirable mensuration AMS concentration.
In addition, present invention also offers the detection method that a kind of AMS detects reagent, comprise the following steps: the reagent R1 prepared and reagent R2 is placed on reagent position corresponding to automatic clinical chemistry analyzer, blank product, standard items and sample is placed at the correspondence position of sample disc, the absorbance change of standard items detection is obtained by reference substance and standard items, again according to the absorbance of the sample of automatic clinical chemistry analyzer mensuration, calculate the AMS concentration of sample.
Further, the temperature of described automatic clinical chemistry analyzer is 35-40 DEG C.
Further, the time delay of described automatic clinical chemistry analyzer is 30-60S, and the reading duration is 120-150S.
Further, the determined wavelength of described automatic clinical chemistry analyzer is respectively predominant wavelength 405nm, commplementary wave length 505nm.
The Cleaning Principle that AMS provided by the invention detects reagent is: using the chloro-4-nitrobenzene-maltotriose of 2-as the substrate of AMS, the maltose terminal molecular of the chloro-4-nitrobenzene-maltotriose of α-amylasehydrolysis 2-, a kind of yellow uitramarine indicator of direct generation, the increase measuring its absorbance at 405nm place is directly proportional to AMS concentration in sample.
In a word, AMS provided by the invention detects reagent, compared with prior art has following advantage:
1) AMS provided by the invention detects reagent not containing sodium azide, can not cause the allosteric of AMS, can reduce the error of measurement result in mensuration process;
2) AMS provided by the invention detects reagent and has stable reagent, do not need to add toolenzyme and time delay short advantage;
3) AMS detection reagent testing result provided by the invention is reproducible, and good stability is a kind of detection reagent of desirable mensuration AMS concentration, is conducive to the clinical practice that this AMS detects reagent.
Accompanying drawing explanation
Fig. 1 is the linear correlation figure of embodiment 2-reagent and GmbH company-reagent.
Embodiment
Further describe the present invention below by way of specific embodiment, the present invention is not limited only to following examples.Within the scope of the invention or not departing from content of the present invention, spirit and scope, the change carried out the present invention, combination or replacement, be apparent for a person skilled in the art, and be included within the scope of the present invention.
embodiment 1,aMS detects reagent
AMS detects reagent and comprises reagent R1 and reagent R2, and described reagent R1 comprises following component:
PH value is MES damping fluid 40mmol/L, NH of 5.5
4cl 80mmol/L, CaCl
25mmol/L, polyglycol 3g/L, p-hydroxybenzoate 2g/L;
Described reagent R2 comprises following component:
PH value is the chloro-4-nitrobenzene-maltotriose 10mmol/L of MES damping fluid 40mmol/L, 2-, the p-hydroxybenzoate 2g/L of 5.5.
embodiment 2,aMS detects reagent
AMS detects reagent and comprises reagent R1 and reagent R2, and described reagent R1 comprises following component:
PH value is MES damping fluid 80mmol/L, NH of 6.0
4cl 120mmol/L, CaCl
210mmol/L, polyglycol 5g/L, p-hydroxybenzoate 6g/L;
Described reagent R2 comprises following component:
PH value is the chloro-4-nitrobenzene-maltotriose 30mmol/L of MES damping fluid 80mmol/L, 2-, the p-hydroxybenzoate 5g/L of 6.0.
embodiment 3,aMS detects reagent
AMS detects reagent and comprises reagent R1 and reagent R2, and described reagent R1 comprises following component:
PH value is MES damping fluid 100mmol/L, NH of 6.5
4cl160mmol/L, CaCl
230mmol/L, polyglycol 10g/L, p-hydroxybenzoate 8g/L;
Described reagent R2 comprises following component:
PH value is the chloro-4-nitrobenzene-maltotriose 80mmol/L of MES damping fluid 100mmol/L, 2-, the p-hydroxybenzoate 8g/L of 6.5.
test example one,aMS detects the correlation test of reagent
1, test reagent: AMS prepared by embodiment 2 detects reagent, the α-determination of amylase reagent of the DiaSysDiagnostic Systems GmbH company of State Food and Drug Administration's accreditation.
2, test method: the AMS utilizing embodiment 2 to prepare detects reagent, the AMS of the DiaSysDiagnostic Systems GmbH company approved with state food pharmaceuticals administration general bureau measures kit (EPS-G7 method), adopt Hitachi 7170 automatic biochemistry analyzer Simultaneously test 20 clinical serum samples to carry out control test, obtain the correlation curve of two kinds of reagent.The parameter of vertical 7170 automatic biochemistry analyzer settings is: temperature is 37 DEG C, and postpone 60s and start read point, the reading duration is about 120s, and determined wavelength is respectively predominant wavelength 405nm, commplementary wave length 505nm.
3, test findings:
As shown in Figure 1, result calculates test findings after measured, and the related coefficient of two kits is 0.9997, and describing both has great correlativity, thus proves that AMS provided by the invention detects reagent and has good practicality.
test example two,aMS detects the stability test of reagent
1, test reagent: AMS prepared by embodiment 2 detects reagent, standard items: Switzerland Roche cfas calibrates serum, quality-control product: the high and low quality-control product provided by Bio-Rad company of the U.S..
2, test method: adopt Hitachi 7170 automatic biochemistry analyzer to measure, the parameter of its setting is: temperature is 37 DEG C, postpone 60s and start read point, the reading duration is about 120s, and determined wavelength is respectively predominant wavelength 405nm, commplementary wave length 505nm.First with standard items calibration, re-use the quality-control product of high and low two levels of AMS detection reagent mensuration prepared by embodiment 2, continue to measure half a year, according to determination data, calculate and test average and the coefficient of variation.
3, test findings
Test findings is as shown in table 1.
The test findings data of the high and low quality-control product of table 1
| Average (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% |
Drawn by table 1, the coefficient of variation that AMS provided by the invention detects the high and low quality-control product that reagent detects is less than 5%, proves that the measurement result of AMS of the present invention detection reagent has good stability.
test example three,aMS detects the replica test of reagent
1, test reagent: AMS prepared by embodiment 2 detects reagent, standard items: Switzerland's Roche just occurs to calibrate serum, sample: the serum collecting clinical high and low level.
2, test method: adopt Hitachi 7170 automatic biochemistry analyzer to measure, the parameter of its setting is: temperature is 37 DEG C, postpone 60s and start read point, the reading duration is about 120s, and determined wavelength is respectively predominant wavelength 405nm, commplementary wave length 505nm.First with standard items calibration, re-use AMS prepared by embodiment 2 and detect the serum 20 times that reagent measures the high and low level of clinical collection, replica test in carrying out batch, according to detection data, calculates test average and the coefficient of variation.
3, test findings
Test findings is as shown in table 2.
The serum test result data of the high and low level of table 2
| Average (U/L) | The coefficient of variation (%) | |
| High level serum | 133.4U/L | 0.72% |
| Low value serum | 24.6U/L | 1.52% |
Drawn by table 2, the coefficient of variation that AMS provided by the invention detects the high and low value serum that reagent detects is less than 5%, proves that the measurement result of AMS of the present invention detection reagent has good repeatability.
Claims (10)
1. AMS detects a reagent, and it is characterized in that, this reagent comprises reagent R1 and reagent R2, and described reagent R1 comprises following component:
MES damping fluid 40-100mmol/L, NH
4cl 80-160mmol/L, CaCl
25-30mmol/L, surfactant 2-12g/L and antiseptic 2-8g/L;
Described reagent R2 comprises following component:
The chloro-4-nitrobenzene-maltotriose 10-80mmol/L and antiseptic 2-8g/L of MES damping fluid 40-100mmol/L, 2-.
2. AMS as claimed in claim 1 detects reagent, and it is characterized in that, this reagent comprises reagent R1 and reagent R2, and described reagent R1 comprises following component:
MES damping fluid 80mmol/L, NH
4cl 120mmol/L, CaCl
210mmol/L, surfactant 5g/L and antiseptic 6g/L;
Described reagent R2 comprises following component:
The chloro-4-nitrobenzene-maltotriose 30mmol/L and antiseptic 5g/L of MES damping fluid 80mmol/L, 2-.
3. AMS as claimed in claim 1 or 2 detects reagent, and it is characterized in that, the pH value of described MES damping fluid is 5.5-6.5.
4. AMS as claimed in claim 3 detects reagent, and it is characterized in that, the pH value of described MES damping fluid is 6.0.
5. AMS as claimed in claim 1 or 2 detects reagent, and it is characterized in that, described surfactant is polyglycol.
6. AMS as claimed in claim 1 or 2 detects reagent, and it is characterized in that, described antiseptic is p-hydroxybenzoate.
7. the AMS as described in as arbitrary in claim 1-6 detects a detection method for reagent, it is characterized in that, comprises the following steps:
The reagent R1 prepared and reagent R2 is placed on reagent position corresponding to automatic clinical chemistry analyzer, blank product, standard items and sample is placed at the correspondence position of sample disc, the absorbance change of standard items detection is obtained by reference substance and standard items, again according to the absorbance of the sample of automatic clinical chemistry analyzer mensuration, calculate the AMS concentration of sample.
8. the detection method of as claimed in claim 7-Amylase detection reagent, it is characterized in that, the temperature of described automatic clinical chemistry analyzer is 35-40 DEG C.
9. the detection method of as claimed in claim 7-Amylase detection reagent, it is characterized in that, the time delay of described automatic clinical chemistry analyzer is 30-60s, and the reading duration is 120-150s.
10. the detection method of as claimed in claim 7-Amylase detection reagent, it is characterized in that, the determined wavelength of described automatic clinical chemistry analyzer is respectively predominant wavelength 405nm, commplementary wave length 505nm.
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| CN108982885A (en) * | 2017-06-05 | 2018-12-11 | 广州东林生物科技有限公司 | The kit of alpha-amylase in a kind of measurement serum |
| 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 |
| CN110951824A (en) * | 2019-11-15 | 2020-04-03 | 中山市创艺生化工程有限公司 | Application of composite buffer system in preparation of α -amylase determination kit |
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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 |
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| CN110951824A (en) * | 2019-11-15 | 2020-04-03 | 中山市创艺生化工程有限公司 | Application of composite buffer system in preparation of α -amylase determination kit |
| 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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