Alpha-amylase detection reagent and detection method
阅读说明：本技术 α-淀粉酶检测试剂及检测方法 (Alpha-amylase detection reagent and detection method ) 是由 钱帅帅 王婷婷 余向东 于 2019-09-18 设计创作，主要内容包括：本发明公开了一种α-淀粉酶检测试剂,化学成分如下：NaCl 50.0～500.0mmol/L,CaCl<Sub>2</Sub>10.0～50.0mmol/L,TRIS-HCL缓冲液50.0～500.0mmol/L,稳定剂EDTA 1.0～5.0mmol/L,叠氮钠0.1～10.0g/L,亚乙基-4-硝基酚-1,4-α-D-麦芽七糖苷0.5～10.0mmol/L,α-葡萄糖苷酶5.0～50.0KU/L；所述缓冲液为N-2羟乙基哌嗪-N’-乙基磺酸缓冲液、TRIS-HCL缓冲液或1,4-哌嗪二乙磺酸缓冲液；所述缓冲液的PH为7.00～8.00。本发明的α-淀粉酶检测试剂取得了如下显著的进步：(1)采用单试剂的配方工艺,减少了仪器试剂位置占用,同时使用更加方便,提高了检测工作效率；(2)特定的配方使得α-淀粉酶检测试剂在开瓶后仍具有较好的稳定性,在开瓶65天后仍未发生变质,可以保证检测结果准确,避免了因开瓶使用后储存过久导致的试剂浪费。(The invention discloses an alpha-amylase detection reagent which comprises the following chemical components of 50.0-500.0 mmol/L of NaCl, 10.0-50.0 mmol/L of CaCl 2 10.0, 50.0-500.0 mmol/L of TRIS-HCL buffer solution, 1.0-5.0 mmol/L of stabilizer EDTA, 0.1-10.0 g/L of sodium azide, 0.5-10.0 mmol/L of ethylene-4-nitrophenol-1, 4-alpha-D-maltoheptaside, and 5.0-50.0 KU/L of alpha-glucosidase, wherein the buffer solution is N-2 hydroxyethyl piperazine-N' -ethyl sulfonic acid buffer solution, TRIS-HCL buffer solution or 1, 4-piperazine diethyl sulfonic acid buffer solution, the pH of the buffer solution is 7.00-8.00, the alpha-amylase detection reagent has the following remarkable improvements, (1) a single-reagent formula is adopted, the occupation of the reagent position is reduced, the use is convenient, the work efficiency is improved, the alpha-amylase detection reagent can be stored more accurately after a bottle is opened, and the starch detection reagent is accurate after a long time is used, and the reagent is prevented from being used.)
1. The alpha-amylase detection reagent is characterized by comprising the following chemical components:
The buffer solution is N-2 hydroxyethyl piperazine-N' -ethyl sulfonic acid buffer solution, TRIS-HCL buffer solution or 1, 4-piperazine diethyl sulfonic acid buffer solution; the pH value of the buffer solution is 7.00-8.00.
2. The reagent for detecting alpha-amylase of claim 1, wherein the stabilizer is MgCl 2 or EDTA.
3. The alpha-amylase detection reagent of claim 1, wherein: the preservative is sodium azide or Proclin300 series.
4. The alpha-amylase assay reagent of claim 1, wherein the chemical composition is as follows:
5. The alpha-amylase detection reagent of claim 4, wherein: the pH of the buffer was 7.5.
6. The alpha-amylase detection reagent of claim 4 or 5, wherein the chemical composition is NaCl300.0mmol/L, CaCl 2 30.0.0 mmol/L, TRIS-HCL buffer 300.0mmol/L, stabilizer EDTA 3.0mmol/L, sodium azide 1.0g/L, ethylene-4-nitrophenol-1, 4-alpha-D-maltoheptaside 5.0mmol/L, alpha-glucosidase 30.0 KU/L.
7. The alpha-amylase detection reagent of claim 4 or 5, wherein the chemical composition is 50.0mmol/L NaCl, 10.0mmol/L CaCl 2 10.0, 50.0mmol/L TRIS-HCL buffer, 1.0mmol/L stabilizer EDTA, 0.1g/L sodium azide, 0.5mmol/L ethylene-4-nitrophenol-1, 4-alpha-D-maltoheptaside, and 5.0KU/L alpha-glucosidase.
8. The alpha-amylase detection reagent of claim 4 or 5, wherein the chemical composition is 500.0mmol/L NaCl, 50.0mmol/L CaCl 2 50.0, 500.0mmol/L TRIS-HCL buffer, 5.0mmol/L stabilizer EDTA, 10.0g/L sodium azide, 10.0mmol/L ethylene-4-nitrophenol-1, 4-alpha-D-maltoheptaside, and 50.0KU/L alpha-glucosidase.
9. a method for detecting an alpha-amylase, which comprises detecting an alpha-amylase using the alpha-amylase detecting reagent according to any one of claims 1 to 7.
Technical Field
The invention relates to an alpha-amylase detection technology, in particular to an alpha-amylase detection reagent and a method for detecting alpha-amylase by using the reagent.
Background
Since amylase is not strongly limited in the size of substrate molecules, but the action site for catalyzing hydrolysis (α -1,4 glycosidic bond in polysaccharide molecules) is very well defined, there are not less than 200 methods for determining amylase that have been developed. The early detection method mainly takes the natural starch as a substrate, and the common defect of the method is that the natural starch lacks uniform and definite molecular structure and chemical property, so that the methodological standardization is difficult to achieve; and because the molecular weight of the natural starch is huge, the concentration which is 10 times Km is difficult to reach when the natural starch is prepared into a substrate solution, the initial rate of enzyme reaction is limited, and the accuracy and the repeatability of determination are poor. In the later period, a method for avoiding the two defects is established, and mainly a method using artificially synthesized malto-oligosaccharide glycoside as a substrate. The method has the advantages that the structure and the molecular mass of the artificially synthesized malto-oligoside are determined, the solubility is good, and higher concentration can be prepared, so that the initial rate of the enzyme reaction reaches the maximum, and the requirement of zero-order reaction is met. The method is suitable for automatic analysis, and has the advantages of high speed, high precision and international expression of enzyme activity.
The alpha-amylase item is a conventional detection item in a hospital at present, the stability of an alpha-amylase detection reagent after opening a bottle is always a focus of attention of the hospital, the stability of the same product cannot be well matched with a buffer solution at present, the reagent can be yellowed due to decomposition of nitrophenol or phenol aniline from a matrix after opening the bottle, the reagent loses reaction and cannot accurately measure a result, and the problems of blank absorbance increase, narrow linear range, poor low-value repeatability and the like are specifically shown, so that the bottle opening stability of the existing product is only 2-3 weeks basically, and the condition of inaccurate result caused by deterioration of the reagent is easy to occur when small-sized hospitals such as community hospitals encounter few patients in non-physical examination time. Therefore, it is necessary to develop a reagent for detecting alpha-amylase with good stability after opening a bottle, so as to facilitate the use of hospitals with small reagent consumption and ensure accurate results.
disclosure of Invention
In order to overcome the defects in the prior art, the application provides the alpha-amylase detection reagent which has the characteristics of good accuracy and good stability after opening the bottle, and avoids waste caused by deterioration of the reagent after long-term storage after the bottle is opened; the detection reagent adopts a single reagent formula process, so that the occupation of instrument reagent positions is reduced, and the detection reagent has the characteristic of convenience in use and improves the detection working efficiency. Correspondingly, the application also provides a method for detecting the alpha-amylase by using the alpha-amylase detection reagent.
For the detection reagent, the technical scheme of the application is as follows:
The alpha-amylase detection reagent comprises the following chemical components:
The buffer solution is N-2 hydroxyethyl piperazine-N' -ethyl sulfonic acid buffer solution, TRIS-HCL buffer solution or 1, 4-piperazine diethyl sulfonic acid buffer solution; the pH value of the buffer solution is 7.00-8.00.
compared with the prior art, the alpha-amylase detection reagent has a specific formula, and the following remarkable improvements are achieved:
(1) by adopting a single reagent formula process, the occupation of instrument reagent positions is reduced, the use is more convenient, and the detection working efficiency is improved;
(2) in the invention, the buffer solution can well relieve the influence of carbon dioxide in the air on a reagent buffer system, so that the damage to the whole reaction environment is relieved, and the proper pH selection of the invention ensures that enzyme substances in the reagent can keep good activity and the preservative with specific concentration, so that the alpha-amylase detection reagent can better inhibit the pollution of sedimentary bacteria in the air, and the alpha-amylase detection reagent still has better stability after the bottle is opened. Tests show that the alpha-amylase detection reagent still does not deteriorate after 65 days of bottle opening, can ensure accurate detection results, and avoids reagent waste caused by long-term storage after bottle opening.
In the foregoing alpha-amylase detection reagent, the stabilizing agent may be magnesium chloride, MgCl2, or chelating agent EDTA.
In the above-mentioned alpha-amylase detection reagent, the preservative may be selected from sodium azide or Proclin300 series.
Preferably, the chemical components of the alpha-amylase detection reagent are as follows:
Tests show that the reagent adopting the formula has good correlation with a contrast reagent, the correlation coefficient can reach more than 0.9995, and the detection accuracy is high.
Preferably, the pH of the buffer is 7.5.
Preferably, the chemical components of the alpha-amylase detection reagent are as follows: NaCl300.0mmol/L, CaCl230.0 mmol/L, TRIS-HCL buffer solution 300.0mmol/L, stabilizer EDTA 3.0mmol/L, sodium azide 1.0g/L, ethylene-4-nitrophenol-1, 4-alpha-D-maltoheptaside 5.0mmol/L, alpha-glucosidase 30.0 KU/L. Experiments show that at the moment, the correlation coefficient of the reagent and the contrast reagent can reach 0.9999, the regression equation is that y is 0.9835x +0.489, and the detection accuracy is high.
preferably, the chemical components of the alpha-amylase detection reagent are as follows: NaCl50.0mmol/L, CaCl210.0 mmol/L, TRIS-HCL buffer 50.0mmol/L, stabilizer EDTA 1.0mmol/L, sodium azide 0.1g/L, ethylene-4-nitrophenol-1, 4-alpha-D-maltoheptaside 0.5mmol/L, alpha-glucosidase 5.0 KU/L. Experiments show that at the moment, the correlation coefficient of the reagent and the contrast reagent can reach 0.9998, the regression equation is that y is 0.9651x +1.0896, and the detection accuracy is high.
Preferably, the chemical components of the alpha-amylase detection reagent are as follows: NaCl500.0mmol/L, CaCl250.0 mmol/L, TRIS-HCL buffer solution 500.0mmol/L, stabilizer EDTA 5.0mmol/L, sodium azide 10.0g/L, ethylene-4-nitrophenol-1, 4-alpha-D-maltoheptaside 10.0mmol/L, alpha-glucosidase 50.0 KU/L. Experiments show that at the moment, the correlation coefficient of the reagent and the contrast reagent can reach 0.9995, the regression equation is that y is 1.0147x-1.3037, and the detection accuracy is high.
For the detection method, the technical scheme of the application is as follows: a method for detecting alpha-amylase, which comprises detecting alpha-amylase by using the alpha-amylase detection reagent of the present application. The detection method has the characteristics of simple operation and convenience while the detection accuracy is guaranteed.
Drawings
FIG. 1 is a graph comparing the correlation of the reagent of example 1 of the present invention with a comparative reagent;
FIG. 2 is a graph comparing the correlation of the agent of example 2 of the present invention with a comparative agent;
FIG. 3 is a graph comparing the correlation of the reagent of example 3 of the present invention with a comparative reagent.
Detailed Description
The invention is further described in the following detailed description (including the examples and figures) without intending to limit the invention thereto.
Detection of alpha-amylase using the alpha-amylase detection reagent of the present invention may employ the alpha-amylase reference method recommended by IFCC.
The alpha-amylase detection reagent is suitable for various full-automatic biochemical analyzers, taking Toshiba TBA-40FR as an example, and the analysis method is shown in tables 1 and 2.
TABLE 1
Sample size: 6ul
The reagent dosage is as follows: 250ul
The determination method comprises the following steps: velocity method
Dominant wavelength: 404nm
secondary wavelength: is free of
A calibration mode: liner
The reaction direction is as follows: is rising
Reaction temperature: 37 deg.C
Reaction time: 1-3 min
TABLE 2
The present invention is further described below by way of examples 1 to 3. The components and concentrations in examples 1-3 are shown in Table 3.
TABLE 3
And (3) correlation test:
The inventive reagent and the contrast reagent (double reagents of Zhejiang century Kangda medical science and technology Co., Ltd., registered label of medical instruments of the people's republic of China is 20182400426), 40 human serums (including normal and abnormal samples) are simultaneously measured according to respective parameters by using a Toshiba 40 full-automatic biochemical analyzer, correlation analysis is performed on the measured values, and the measurement is performed according to the parameters in tables 1 and 2, as shown in FIGS. 1, 2 and 3, the X axis is the measured value of the contrast reagent, the Y axis is the measured value of the inventive reagent (AMY unit U/L), as can be seen from FIGS. 1, 2 and 3, the correlation coefficients of the two reagents are respectively R 2 0.9999, R 2 0.9998, R 2 is 0.9995, the regression equations are respectively Y0.9835X +0.489, Y0.9651X +1.0896, Y1.0147X-1.3037, the results show that the inventive reagent and the inventive serum has good correlation detection accuracy.
The above experiment was carried out using a TBA-40FR full-automatic biochemical analyzer manufactured by Toshiba, however, the reagent of the present invention is not limited to the above-mentioned apparatus, and is also applicable to other full-automatic or semi-automatic biochemical analyzers.
Bottle opening stability test:
the purpose of this test was to monitor the stability of the reagent at the time of opening, and the test subjects were the detection reagents of examples 1 to 3 of the present invention. And matching with third-party Landau calibration products and quality control products.
The instrument comprises the following steps: toshiba TBA-40FR full-automatic biochemical analyzer.
The method comprises the following operation steps: the quality control was measured 3 times, once every 3 to 7 days, using calibrator.
Test protocol: the reagent is placed in a reagent chamber after being unpacked, and the deviation between the measured value of the RANDOX composite quality control serum and the target value thereof is regularly monitored by using the first calibration data.
And (4) analyzing results: the test quality control value and the quality control deviation are required to be less than or equal to 10 percent.
The following table 4-6 shows the stability test results after opening the bottle for 65 days. The data in tables 4-6 show that the quality control deviation of the reagent is still less than or equal to 10% at the 65 th day of bottle opening, so that the bottle opening stability of the reagent is enough to meet the daily use of the reagent in hospitals as small as community hospitals, and the condition that the reagent is unstable in bottle opening and goes bad due to small daily use amount of the reagent is not needed to be worried about.
Table 4: EXAMPLE 1 bottle opening stability test
Time of opening bottle
Day 0
Day 4
Day 7
Day 10
Day 17
Day 24
Day 31
Quality control L (mean value)
82.1
83.8
82.3
83.5
83.0
86.35
81.7
Relative deviation%
-3.4
-1.4
-3.2
-1.8
-2.3
1.6
-3.9
Quality control H (mean value)
270.7
268.05
264
270.25
272.1
270.3
271.5
Relative deviation%
3.3
2.3
0.8
3.1
3.8
3.2
3.6
Time of opening bottle
Day 38
Day 43
Day 48
Day 53
Day 58
Day 65
Quality control L (mean value)
88.0
84.65
85.0
84.3
85.2
83.7
Relative deviation%
3.6
-0.4
0.0
-0.8
0.2
-1.5
Quality control H (mean value)
273.7
270.55
267.0
267.9
262
265.3
Relative deviation%
4.5
3.3
1.9
2.3
0.0
1.3
Table 5: EXAMPLE 2 decap stability test
Time of opening bottle
Day 0
Day 4
Day 7
Day 10
Day 17
Day 24
Day 31
Quality control L (all are)Value)
83.9
85.1
85.4
85.3
82.0
83.2
82.8
Relative deviation%
-1.3
0.1
0.5
0.4
-3.5
-2.1
-2.6
Quality control H (mean value)
282.6
279.1
278.4
275.3
273.6
270.9
274.2
Relative deviation%
7.9
6.5
6.3
5.1
4.4
3.4
4.7
Time of opening bottle
Day 38
Day 43
Day 48
Day 53
Day 58
Day 65
Quality control L (mean value)
87.4
84.8
84.7
85.2
88.0
84.2
Relative deviation%
2.8
-0.3
-0.4
0.2
3.5
-0.9
Quality control H (mean value)
274.7
267.9
270.1
262
266.3
268.7
Relative deviation%
4.8
2.3
3.3
0.0
1.6
2.6
Table 6: example 3 bottle opening stability test
Time of opening bottle
Day 0
Day 4
Day 7
Day 10
Day 17
Day 24
Day 31
Quality control L (mean value)
84.2
83.5
83.1
83.6
85.3
81.8
82.7
Relative deviation%
-0.9
-1.8
-2.3
-1.6
0.4
-3.8
-2.7
Quality control H (mean value)
268.7
266.7
264.7
264.4
265.8
269.5
274.5
relative deviation%
2.6
1.9
1.0
0.9
1.5
2.9
4.8
Time of opening bottle
Day 38
Day 43
Day 48
Day 53
Day 58
Day 65
Quality control L (mean value)
81.9
80.7
82.0
81.2
80.3
81.4
Relative deviation%
-3.6
-5.1
-3.5
-4.5
-5.5
-4.2
Quality control H (mean value)
270.8
269.1
265.6
262.2
260.5
259.3
Relative deviation%
3.4
2.7
1.4
0.1
-0.6
-1.0
The above general description of the invention and the description of the specific embodiments thereof, as referred to in this application, should not be construed as limiting the technical solutions of the invention. Those skilled in the art can add, reduce or combine the technical features disclosed in the general description and/or the specific embodiments (including the examples) to form other technical solutions within the protection scope of the present application according to the disclosure of the present application without departing from the structural elements of the present invention.
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