CN102288559B - Method and kit for detecting amylase - Google Patents

Abstract

The invention relates to the technical field of medical detection, and discloses a method and a kit for detecting amylase. In the method for detecting amylase provided by the present invention, in a buffer solution of pH 7.2, the sample to be tested is mixed with soluble starch to obtain a mixed solution, potassium iodide is mixed with acidic potassium iodate to react with the obtained mixed solution, and the absorbance is detected at a wavelength of 630nm , calculate the concentration of amylase in the sample to be tested according to the absorbance of the blank tube without enzymatic catalysis after the same treatment as above. In the detection method of the present invention, potassium iodide and potassium iodate generate iodine under acidic conditions, and combine with unreacted starch to form a blue complex, avoiding the direct use of iodine liquid, which is easy to sublime and volatilize, causing the reduction and lightening of components and affecting the color development of the reaction, and improving detection. Result accuracy. The kit of the invention has good stability, long storage period, wide application range, and is easy to popularize and use, and can be applied to hospitals at all levels, health departments and medical and biological scientific research units to measure the amylase content in serum or urine.

Description

A method and kit for detecting amylase

technical field

The invention relates to the technical field of medical testing and determination, in particular to a method and kit for detecting amylase.

Background technique

Amylase (α-Amylase), referred to as AMS or AMY, is a type of enzyme that can hydrolyze starch molecules. It generally acts on α-1,4-glucans such as soluble starch, amylose, and glycogen to hydrolyze α-1, 4-Glycosidic bonds, resulting in dextran, maltose and glucose molecules. Amylase is abundant in the pancreas and salivary glands. Pancreatic amylase is excreted into the digestive tract in an active state by the pancreas. It is the most important enzyme that hydrolyzes carbohydrates. It belongs to α-amylase like the amylase secreted by salivary glands. Amylase has a small molecular weight and can be filtered through the glomerulus and excreted with urine. When suffering from pancreatic diseases or pancreatic exocrine dysfunction, amylase activity can be increased or decreased. Therefore, the determination of amylase in serum and urine is of great significance for the diagnosis of pancreatitis. Urinary amylase levels fluctuate greatly, so serum amylase detection is preferred, or both are measured at the same time.

There are many methods for the determination of amylase activity in serum and urine, mainly viscometry, turbidimetry, saccharification, iodine-starch colorimetry, enzyme rate method and dye release method. Among them, viscometry and turbidimetry have been eliminated due to many influencing factors and lack of specificity and sensitivity. Although the saccharification method can directly measure enzyme activity, the operation is complicated and not suitable for routine clinical examination. At present, the most widely used methods are iodometric method, enzyme rate method and dye release method. Among them, the iodine-starch colorimetric method has been widely used clinically because of its low cost, no need for expensive instruments, stable reagents, easy operation, and reliable results.

The reaction principle of the iodine-starch colorimetric method for the determination of amylase in serum and urine is that α-amylase in serum (syrup) and urine catalyzes the hydrolysis of α-1,4 glycosidic bonds in starch molecules to produce glucose, maltose and Alpha-1,6 glycosidic bond branched dextrin. Under the condition of quantitative substrate, iodine solution is added after the reaction to combine with unhydrolyzed starch to form a blue complex. Compared with the blank tube without enzymatic reaction, the greater the amylase activity, the greater the remaining starch. The less it is, the lighter the color of the reaction solution is. Place the final product under a UV/visible light analyzer or a semi-automatic biochemical analyzer, and detect the absorbance value at the main wavelength of 630nm, so as to measure the amylase content in the sample.

However, the iodine solution of the color developer in the above method is easy to sublimate and volatilize, causing the components to decrease and become light, which affects the color development of the reaction, resulting in a decrease in the accuracy of the detection result.

Contents of the invention

In view of this, the purpose of the present invention is to provide a kit for detecting amylase with high stability and high accuracy.

For realizing the purpose of the present invention, the present invention adopts following technical scheme:

A method for detecting amylase, comprising:

Step 1. In a buffer solution of pH 7.2, the sample to be tested is mixed with soluble starch to obtain a mixed solution;

Step 2, potassium iodide is mixed with acidic potassium iodate, reacted with the mixed solution obtained in step 1, and detects the absorbance at a wavelength of 630nm, and calculates the amylase in the sample to be tested according to the absorbance of the blank tube that has not been enzymatically catalyzed through the same treatment as above concentration.

α-amylase in serum (syrup) and urine catalyzes the hydrolysis of α-1,4 glycosidic bonds in starch molecules to produce glucose, maltose and dextrins containing α-1,6 glycosidic bond branches. Under the condition of substrate quantification, after the reaction, iodine solution is added to combine with unhydrolyzed starch to form a blue complex, and the depth of blue is compared with the blank tube without enzymatic reaction, and the absorbance at the main wavelength of 630nm is detected. Value size, so as to measure the content of amylase in the sample. The detection method of the present invention uses potassium iodide and acidic potassium iodide as the chromogenic liquid, and potassium iodide and potassium iodate generate iodine under acidic conditions, and combine with unreacted starch to form a blue complex, avoiding the direct use of iodine liquid which is easy to sublimate and volatilize to form components Reduce the effect of lightening on the color development of the reaction, and improve the accuracy of the test results.

Preferably, the molar ratio of potassium iodate to potassium iodide is 1:5-6.

Preferably, the acidic potassium iodate is 8-12 mmol/L potassium iodate solution with pH 2.0-3.0.

In order for the enzyme to bind well to the substrate, both the enzyme and the substrate need a buffer to provide a suitable dissociation environment. The ionic strength of the buffer also affects the activity of the enzyme. If the ionic strength is too high, the electrolyte will interfere with the combination of the enzyme and the substrate, and the enzyme activity will gradually decrease. However, if the ionic strength is too low, the enzyme activity may not be activated. Generally, the ionic strength close to that of the body fluid in the physiological environment is selected. Therefore, in order to provide a suitable environment for enzymatic hydrolysis in the detection method of the present invention, the sample to be tested is mixed with soluble starch in a buffer solution with a pH of 7.2. The buffer at pH 7.2 includes: disodium hydrogen phosphate-citric acid buffer, Tris-hydrochloric acid buffer and phosphate buffer. Preferably, the buffer solution of pH7.2 in the detection method of the present invention is a phosphate buffer solution of 50-100mmol/L pH7.2, including disodium hydrogen phosphate-sodium dihydrogen phosphate buffer and disodium hydrogen phosphate - Potassium dihydrogen phosphate buffer.

Preferably, the soluble starch concentration is 960mg/L.

The present invention also provides a kit for detecting amylase, comprising 960mg/L soluble starch, 50-100mmol/L pH7.2 phosphate buffer, 8-10mmol/L pH2.0-3.0 potassium iodate solution and 30-50mmol/L potassium iodide.

Preferably, the kit of the present invention further includes 50-70 mmol/L of sodium benzoate, and sodium benzoate is used as a preservative to ensure the stability of the reagent.

The test kit for detecting amylase provided by the present invention can be two reagents, the phosphate buffer saline and potassium iodide of soluble starch and pH7.2 are made into the first reagent, and acidic potassium iodate is made into the second reagent. When the kit detects amylase, the first reagent is mixed with the second reagent, so as to detect the amylase in the sample to be tested. The test kit for detecting amylase provided by the present invention can also be a multi-reagent, the first reagent is made of soluble starch and pH7.2 phosphate buffer, the second reagent is made of acidic potassium iodate, and the second reagent is made of potassium iodide. The third reagent, when detecting amylase, mix the first reagent with the second reagent and the third reagent to detect amylase in the sample to be tested. Due to the poor stability of the aqueous solution of soluble starch, the kit of the present invention makes the reagent containing soluble starch into a dry powder reagent, which is dissolved before use.

As can be seen from the above-mentioned technical scheme, the detection method of the present invention uses potassium iodide and acidic potassium iodide as the chromogenic solution, and potassium iodide and potassium iodate generate iodine under acidic conditions, which are combined with unreacted starch to form a blue complex to avoid Direct use of iodine solution is easy to sublimate and volatilize, resulting in the reduction and lightening of components, which affects the reaction and color development, and improves the accuracy of test results. The test shows that the detection method of the present invention has higher accuracy and precision, and the same sample is repeatedly detected, and the standard deviation rate (variation coefficient) between each result is 1.92%, which is less than 3% of the standard, and linear Wide range, the linear range of amylase in serum can reach 800U/L, and the linear range of amylase in urine can reach 1600U/L. The kit of the invention has good stability, long storage period, wide application range, and is easy to popularize and use, and can be applied to hospitals at all levels, health prevention departments and medical and biological scientific research units to measure the amylase content in serum or urine.

Detailed ways

The embodiment of the invention discloses a method and a kit for detecting amylase. Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The products and methods of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention Invent technology.

In order to further understand the present invention, the present invention will be described in detail below in conjunction with examples.

Embodiment 1: A kit for detecting amylase according to the present invention.

The kit for detecting amylase in the present invention is multi-reagent.

The first reagent is a dry powder reagent, including 960mg/L soluble starch, 50mmol/L pH7.2 phosphate buffer and 60mmol/L sodium benzoate;

The second reagent is 30mmol/L potassium iodide;

The third reagent is 8 mmol/L potassium iodate solution at pH 2.0.

Embodiment 2: A kit for detecting amylase according to the present invention.

The kit for detecting amylase in the present invention is multi-reagent.

The first reagent is a dry powder reagent, including 960mg/L soluble starch, 20mmol/L pH7.2 phosphate buffer and 50mmol/L sodium benzoate;

The second reagent is 50mmol/L potassium iodide;

The third reagent is 12mmol/L potassium iodate solution at pH3.0.

Embodiment 3: A kit for detecting amylase according to the present invention.

The kit for detecting amylase of the present invention is a double reagent.

The first reagent is a dry powder reagent, including 960mg/L soluble starch, 100mmol/L pH7.2 phosphate buffer, 48mmol/L potassium iodide and 70mmol/L sodium benzoate;

The second reagent is 8 mmol/L potassium iodate solution at pH 2.5.

Example 4: Method for detecting amylase.

Set the reaction temperature of the semi-automatic biochemical analyzer at 37°C, the reaction method is the end point method, the main wavelength of the measurement is 630nm, and the reaction direction is negative reaction. Take the sample to be tested and mix it with 960mg/L soluble starch, incubate for 360s, then add 40mmol/L potassium iodide and 8mmol/LpH2.0 acidic potassium iodate mixed solution, mix evenly, place it in a semi-automatic biochemical analyzer, detect and record at a wavelength of 630nm of absorbance. Blank tubes that were not enzymatically treated were the same as above. Calculate the concentration of amylase in the sample to be tested according to the calculation formula C _sample =(A _blank -A _sample )/A _blank ×(C×V)/(t×V _sample ), wherein, C _sample is the sample concentration to be tested; A _Sample is the absorbance of the sample; A _blank is the absorbance of the blank tube; C is the concentration of soluble starch; V is the volume of soluble starch; t is the reaction time (min); V _sample is the volume of the sample to be tested. Since the international unit stipulates that 4 mg of starch is hydrolyzed per liter of sample for 1 minute to be 1 international unit (U/L), so in order to convert the obtained amylase concentration into an international unit, it is necessary to use the formula C _{sample (international unit)} = C _sample × (1 × 1000)/4, where 1 is the reaction time (min) specified by the international unit; 1000 is the factor for converting mL into L; 4 is the amount of hydrolyzed starch per liter of sample specified by the international unit.

Embodiment 5: Analysis of the accuracy of the detection method of the present invention

20 clinical samples were taken, and the amylase content in the samples was detected by using the CB171 semi-automatic biochemical analyzer according to the method described in Example 4 and the Gal-G ₂ -CNP substrate method respectively. The results are shown in Table 1.

Table 1 Test results of amylase content in samples

It can be seen from the results in Table 1 that compared with the existing Gal-G ₂ -CNP substrate method, the detection results of the detection method of the present invention are similar, with a correlation coefficient of 0.995, and the detection results of the detection method of the present invention are accurate and reliable.

Embodiment 6: Repeatability detection of detection method of the present invention

Taking the routine serum sample as the sample to be tested, according to the method described in Example 4, using the CB171 semi-automatic biochemical analyzer to detect the amylase content in the sample to be tested, compared with the existing Gal-G ₂ -CNP substrate method, the results are shown in Table 2.

Table 2 Test results of sample repeatability

The 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%

As can be seen from the results in Table 2, the coefficient of variation CV=1.92% for amylase detected by the detection method of the present invention is less than 5%, which meets the "General Requirements for In Vitro Diagnostic Reagents" and is smaller than the existing Gal- _G2 -CNP base The coefficient of variation of the physical method shows that the detection method of the present invention has good repeatability.

Embodiment 7: the detection of method linearity of the present invention

Take high-value serum with an amylase concentration close to 800U/L, dilute it into 5 different concentration gradients, and the theoretical concentration values are 50, 100, 200, 400, 800U/L in sequence. According to the method described in Example 4, use CB171 semi-automatic The biochemical analyzer detects the amylase in the sample to be tested. Each concentration is measured twice, and the average value is taken. The correlation coefficient r value is calculated according to the formula. The results are shown in Table 3.

Take high-value urine with an amylase concentration close to 1600U/L, dilute it into 5 different concentration gradients, and the theoretical concentration values are 100, 200, 400, 800, 1600U/L in sequence. According to the method described in Example 4, use CB171 The amylase in the sample to be tested was detected by a semi-automatic biochemical analyzer, each concentration was measured twice, the average value was taken, and the correlation coefficient r value was calculated according to the formula. The results are shown in Table 4.

Table 3 Linearity detection results of serum samples

Table 4 Linearity detection results of urine samples

As can be seen from the results of Table 3 and Table 4, the linear range of amylase in the detection method of the present invention can reach 800U/L, and the linear range of amylase in urine can reach 1600U/L, showing that the detection method of the present invention Wide linear range and wide application range.

The descriptions of the above embodiments are only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (5)

1. A method for detecting amylase, characterized in that, comprising: Step 1. In a buffer solution of pH 7.2, the sample to be tested is mixed with soluble starch to obtain a mixed solution; Step 2, potassium iodide is mixed with acidic potassium iodate, reacted with the mixed solution obtained in step 1, and detects the absorbance at a wavelength of 630nm, and calculates the amylase in the sample to be tested according to the absorbance of the blank tube that has not been enzymatically catalyzed through the same treatment as above concentration; Wherein, the soluble starch concentration is 960mg/L; The buffer solution of described pH7.2 is the phosphate buffer saline of 50～100mmol/L pH7.2; The 50-100mmol/L pH7.2 phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer or disodium hydrogen phosphate-potassium dihydrogen phosphate buffer. 2. detection method according to claim 1, is characterized in that, the mol ratio of described potassium iodate and potassium iodide is 1:4～6. 3. detection method according to claim 1, is characterized in that, described acid potassium iodate is the potassium iodate solution of 8～12mmol/LpH2.0～3.0. 4. A kit for detecting amylase, characterized in that it consists of: 960mg/L soluble starch, 50-100mmol/L pH7.2 phosphate buffer, 8-12mmol/L pH2.0-3.0 iodic acid Potassium and 30-50mmol/L potassium iodide; The 50-100mmol/L pH7.2 phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer or disodium hydrogen phosphate-potassium dihydrogen phosphate buffer. 5. The kit according to claim 4, further comprising 50-70 mmol/L of sodium benzoate.