Alpha-amylase determination reagent and kit

Abstract

The alpha-amylase determination reagent provided by the invention firstly comprises a reagent I, the reagent I is composed of a buffer solution, a surfactant, a preservative, fibrin and 2-chloro-4-nitrobenzene-maltotriose, and the pH value of the solution of the reagent I is 6.0-7.0. According to the present invention, the fibrous protein is added, such that the fibrous protein can stably exist in the reagent; and the fibrin has good solubility in a buffer solution and stable properties. Furthermore, the fibrin can well form a stable complex with copper ions in a detection sample, so that the freeness of the ions is reduced, a relatively stable complex is formed, and when an alpha-amylase reaction is performed, the interference effect of the ions is reduced, and a better detection purpose is achieved.

Description

Technical Field

[0001] The invention relates to a detection kit, in particular to an alpha-amylase determination reagent and a kit capable of reducing partial ion interference in a sample and improving detection accuracy. Background Art

[0002] α-amylase is an important enzyme that is mainly responsible for hydrolyzing α-1,4-glycosidic bonds in starch and glycogen. In medicine, the detection of α-amylase is often used to evaluate pancreatic function and diagnose related diseases, such as acute pancreatitis, chronic pancreatitis, pancreatic tumors, etc.

[0003] In the prior art, 2-chloro-4-nitrophenylmaltotriose can be used as a substrate to detect the activity of α-amylase. The basic principle of this detection is to monitor the generated product through enzymatic reaction, thereby reflecting the activity of the enzyme. The reaction product can be detected by UV / visible spectrophotometry, which has characteristic absorption at a specific wavelength and can be quantified by measuring absorbance. The product generated by the reaction will increase over time, and the activity of α-amylase can be obtained based on the change in absorbance.

[0004] Research has found that the presence of some heavy metal ions in blood samples can easily lead to distortion of test results. This is because the high concentration of metal ions combines with α-amylase to cause activity inhibition and poisoning, which will lead to distortion of test results. For example, for patients with common high serum copper concentrations, the test results will be distorted.

[0005] Therefore, for test samples with high serum copper concentrations, it is of positive significance to inhibit serum copper and reduce this effect. Summary of the invention

[0006] In order to solve the problems existing in the above-mentioned technologies, the present invention provides a technology for inhibiting the interference of serum copper in α-amylase determination.

[0007] The present invention first provides an alpha-amylase assay reagent, which comprises a reagent I, wherein the reagent I is composed of a buffer, a surfactant, a preservative, fibrin and 2-chloro-4-nitrobenzene-maltotriose, and the pH of the reagent I solution is 6.0 to 7.0.

[0008] Preferably, the buffer is a phosphate buffer, and the buffer concentration is 0.1 mol / L to 1 mol / L; the surfactant is polyethylene glycol, and the surfactant concentration is 1 g / L to 5 g / L; the preservative is a paraben, and the preservative concentration is 5 g / L to 10 g / L.

[0009] Preferably, the pH of the reagent I solution is 6.0 to 6.5.

[0010] Preferably, the concentration of the fibrin is 10 μmol / L to 100 μmol / L. The concentration of the fibrin can be appropriately adjusted according to the concentration of copper ions in serum copper to achieve the maximum inhibitory effect.

[0011] The present invention also provides an alpha-amylase assay kit, which also includes a reagent II for control. The reagent II is composed of a buffer, a surfactant, a preservative and fibrin. The dosage ratio of the substances in the reagent II is the same as that of the reagent I.

[0012] After the reagent I is mixed with the sample for reaction, it is placed in the fully automatic biochemical analyzer as a sample to be tested;

[0013] Reagent II is used as a blank reference substance; and a standard curve is provided;

[0014] Obtain the absorbance of the sample to be tested, and determine the corresponding α-amylase concentration after calculation.

[0015] The detection temperature is 36°C to 38°C; the detection wavelengths are 405nm for the main wavelength and 505nm for the secondary wavelength.

[0016] The α-amylase assay kit of the present invention can stably exist in the reagent by adding fibrin, and the fibrin has good solubility in the buffer and stable properties. Furthermore, the fibrin can well form a stable complex with the copper ions in the test sample, so that the freeness of these ions is reduced and a relatively stable complex is formed. In this way, when the α-amylase reaction is carried out, the interference of these ions is reduced, and a better detection purpose is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the coordination of the fibrin of the present invention and copper ions. DETAILED DESCRIPTION

[0018] The present invention first provides an alpha-amylase assay reagent, which comprises a reagent I, wherein the reagent I is composed of a buffer, a surfactant, a preservative, fibrin and 2-chloro-4-nitrobenzene-maltotriose, and the pH of the reagent I solution is 6.0 to 7.0.

[0019] Preferably, the buffer is a phosphate buffer, and the buffer concentration is 0.1 mol / L to 1 mol / L; the surfactant is polyethylene glycol, and the surfactant concentration is 1 g / L to 5 g / L; the preservative is a paraben, and the preservative concentration is 5 g / L to 10 g / L. The above ranges all include endpoint values.

[0020] Preferably, the pH of the reagent I solution is 6.0 to 6.5.

[0021] Preferably, the concentration of the fibrin is 10 μmol / L to 100 μmol / L.

[0022] The present invention also provides an α-amylase assay kit, further comprising a reagent II for control, wherein the reagent II is composed of a buffer, a surfactant, a preservative and fibrin, and the dosage ratio of the substances in the reagent II is the same as that of the reagent I. When performing instrument detection, the sample test is performed by the reagent I, and the reagent II is used as a blank control to facilitate and quickly obtain the data of the sample detection, and the corresponding concentration is calculated according to the linear formula of the standard. The entire instrument use process and the concentration calculation process belong to the prior art and will not be described in detail here.

[0023] After the reagent I is mixed with the sample for reaction, it is placed in the fully automatic biochemical analyzer as a sample to be tested;

[0024] Reagent II is used as a blank reference substance; and a standard curve is provided;

[0025] Obtain the absorbance of the sample to be tested, and determine the corresponding α-amylase concentration after calculation.

[0026] The detection temperature is 36°C to 38°C; the detection wavelengths are 405nm for the main wavelength and 505nm for the secondary wavelength.

[0027] The α-amylase assay kit of the present invention can stably exist in the reagent by adding fibrin, and the fibrin has good solubility in the buffer and stable properties. Furthermore, the fibrin can well form a complex with the copper ions in the test sample, specifically Figure 1 The schematic diagram is shown. This reduces the freeness of these ions and forms a more stable complex. When the α-amylase reaction is carried out, the interference of these ions is reduced, achieving a better detection purpose.

[0028] Example: The buffer is selected as phosphate buffer, the buffer concentration is 0.1 mol / L; the surfactant is polyethylene glycol, the surfactant concentration is 1 g / L; the preservative is paraben, the preservative concentration is 5 g / L, and the concentration of 2-chloro-4-nitrobenzene-maltotriose is 10 mmol / L, i.e. 0.01 mol / L. Then different concentrations of fibrin are given to detect the accuracy of the change of the detection value under different concentrations of fibrin content.

[0029] Table 1 shows the changes in the accuracy of serum copper at different fibrin content levels from 10 μmol / L to 100 μmol / L.

[0030]

[0031] It can be seen from the table above that abnormal serum copper (serum copper far exceeds the normal range of healthy people) will lead to distortion of the detection concentration; specifically, in the control example, no fibrin was added, and the standard concentration was 100U / L, but the test result was 92U / L, which means that the abnormal serum copper led to reduced α-amylase activity or poisoning, so the test result was low; when the fibrin concentration was gradually increased, the accuracy of the detection concentration gradually increased, and when the fibrin concentration reached 30μmol / L, the accuracy of the test result was guaranteed.

[0032] It should be noted that the number of test samples in each embodiment or control example in Table 1 above is greater than 5 groups, the average value is taken as the detection concentration (mean), and the R variance correlation is greater than 0.99.

[0033] When testing, the standard is a pre-configured reagent with a required concentration of serum copper. Then the test is performed using the reagent I of the present invention, and the reagent II is used as a blank control.

Claims

1. An α-amylase assay reagent, characterized in that: The invention comprises a reagent I, wherein the reagent I is composed of a buffer, a surfactant, a preservative, fibrin and 2-chloro-4-nitrobenzene-maltotriose, and the pH value of the reagent I solution is 6.0 to 7.

0.

2. The α-amylase assay reagent according to claim 1, characterized in that The buffer is a phosphate buffer, and the buffer concentration is 0.1 mol / L to 1 mol / L; the surfactant is polyethylene glycol, and the surfactant concentration is 1 g / L to 5 g / L; the preservative is a paraben, and the preservative concentration is 5 g / L to 10 g / L.

3. The α-amylase assay reagent according to claim 1, characterized in that The pH of the Reagent I solution is 6.0 to 6.

5.

4. The α-amylase assay reagent according to claim 1, characterized in that The concentration of the fibrin is 10 μmol / L to 100 μmol / L.

5. The α-amylase assay reagent according to claim 4, characterized in that Fibrinogen is 30μmol / L.

6. An α-amylase assay kit, characterized in that: The invention comprises the α-amylase assay reagent according to any one of claims 1 to 4, and also comprises a reagent II for control, wherein the reagent II comprises: a buffer, a surfactant, a preservative and fibrin, and the dosage ratio of the substances in the reagent II is the same as that of the reagent I.

7. The α-amylase assay kit according to claim 6, characterized in that After the reagent I is mixed with the sample for reaction, it is placed in the fully automatic biochemical analyzer as a sample to be tested; Reagent II is used as a blank reference; and a standard curve is provided; Obtain the absorbance of the sample to be tested, and determine the corresponding α-amylase concentration after calculation.

8. The α-amylase assay kit according to claim 6, characterized in that The detection temperature is 36°C to 38°C; the detection wavelengths are 405nm for the main wavelength and 505nm for the secondary wavelength.

Images