CN108982885B

CN108982885B - Kit for measuring alpha-amylase in serum

Info

Publication number: CN108982885B
Application number: CN201710412228.5A
Authority: CN
Inventors: 张传春; 屈兴翠; 白丽娟; 谭再丽; 罗余香
Original assignee: Guangzhou Donglin Biotechnology Co ltd
Current assignee: Guangzhou Donglin Biotechnology Co ltd
Priority date: 2017-06-05
Filing date: 2017-06-05
Publication date: 2021-11-16
Anticipated expiration: 2037-06-05
Also published as: CN108982885A

Abstract

The invention provides a determination kit for determining alpha-amylase in serum, which is a liquid double reagent. The reagent comprises a reagent 1(R1) and a reagent 2(R2), wherein R1 mainly comprises hydroxyethyl piperazine ethanethiosulfonic acid (HEPES), alpha-glucosidase, sodium chloride, calcium chloride and cerium nitrate; r2 mainly comprises 4, 6-ethylene-4-nitrophenol-alpha-heptose (E-PNP-G7) and cerium nitrate. The cerium nitrate is added into the kit for measuring the alpha-amylase in the serum, so that the performance of the kit is further improved, and the reactivity of the alpha-amylase is improved.

Description

Kit for measuring alpha-amylase in serum

Technical Field

The invention belongs to the technical field of medical in-vitro diagnosis, and particularly relates to a kit for determining alpha-amylase in serum.

Background

The pancreas contains a large number of digestive enzymes, and in clinical work, the most prominent are 3 pancreatic enzymes, namely trypsin, lipase and amylase. In the case of pancreatic dysfunction, on the one hand, secretion dysfunction causes a decrease in the amount of enzymes excreted into the duodenum, and on the other hand, the amount of enzymes escaping into the blood increases, increasing the amount of enzymes in the blood and urine. Depending on the type of isomerism of the amylase hydrolysate, alpha-amylases are present in animals and microorganisms, and beta-amylases are mainly found in higher plants, but have also been reported to be present in bacteria, milk, and molds.

Alpha-amylase is valuable in the diagnosis of not only pancreatic diseases, non-pancreatic diseases, and salivary gland diseases, but also an important index in the diagnosis of abdominal diseases, giant amylase anemia, and the like. A more common use in clinical testing is the serum alpha-amylase assay.

So far, more than 200 methods for measuring alpha-amylase are available, mainly comprising:

1. method for measuring total alpha-amylase activity by using starch as substrate

There are a distinction between viscometry, turbidimetry and iodine-starch coloration, of which viscometry has been stopped since long due to technical difficulties, poor accuracy and precision. The turbidimetric assay has the disadvantage that the substrate is unstable and difficult to label. The iodine-starch color generation method is a semi-quantitative method, cannot accurately reflect the enzyme activity, has low sensitivity and is easy to cause clinical missed diagnosis. The method is eliminated by the Ministry of health in China.

2. Dry chemical assay for alpha-amylase

The method has the characteristics of quick bedside measurement, simplicity, accuracy, precision, practicality and the like, is suitable for clinical application, but is difficult to popularize due to a plurality of factors such as low cognitive degree, high instrument price and reagent price and the like.

3. Alpha-amylase activity determination method using malto-oligosaccharide as substrate

Including direct assays and enzyme-coupled assays using malto-oligosaccharides as substrates. Among them, the direct measurement method has a disadvantage of environmental pollution. The enzyme coupling assay method using maltooligosaccharide as a substrate comprises an enzyme coupling assay method using maltopentose as a substrate, maltotetraose as a substrate and maltoheptaose as a substrate, wherein the method using maltopentose as a substrate has more applied tool enzymes and hemolysis has a larger influence on the assay result; the reaction is interfered by taking maltotetraose as an intermediate product of endogenous maltose, glucose and the like in a substrate method; the maltoheptaose is a substrate method, has the advantages that the hydrolysis rate is increased, the affinity of the maltoheptaose to alpha-amylase isozyme is closest to that of starch, the method has the problem that the reactivity of amylase and the substrate can be changed due to chemical modification, the method cannot completely meet the reference method standard established by IFCC, and the method is closest to the standard compared with other methods.

Therefore, the invention researches an enzyme coupling determination method by improving the method of taking maltoheptaose as a substrate, solves the problem of reactivity of amylase and the substrate, ensures that the amylase and the substrate meet the standard of a reference method established by IFCC as much as possible, and can solve the current clinical application problem.

Disclosure of Invention

In order to overcome the defects of the prior art, although the patent 201410401270.3 adds sodium dodecyl benzene sulfonate into the kit to improve the performance of the kit, the inventor of the present invention finds through experiments that cerium nitrate can better improve the performance of the amylase detection kit, improve the detection sensitivity, and further reduce the minimum detection limit concentration, so that the cerium nitrate is added in the present invention to further improve the performance of the alpha-amylase detection kit.

Detailed description of the preferred embodiments

Specifically, the invention relates to the following aspects, and more specifically, the invention relates to a serum alpha-amylase determination kit, which is a liquid double reagent and consists of a reagent 1(R1) and a reagent 2(R2), wherein the R1 comprises the following components:

the R2 comprises the following components:

0.1-10 mmol/L of 4, 6-ethylene-4-nitrophenol-alpha-heptose (E-PNP-G7)

0.1-1 mmol/L of cerium nitrate

Preferably, the R1 comprises the following components:

the R2 comprises the following components:

E-PNP-G7 5mmol/L

cerium nitrate 0.5mmol/L

Detailed Description

The present invention is described in detail below with reference to specific examples, but the use and purpose of these exemplary embodiments are merely to exemplify the present invention, and do not set forth any limitation on the actual scope of the present invention in any form, and the scope of the present invention is not limited thereto.

Example 1

The kit is a liquid double reagent and consists of a reagent 1(R1) and a reagent 2(R2), wherein the R1 comprises the following components:

the R2 component is:

E-PNP-G7 5mmol/L

cerium nitrate 0.5mmol/L

Example 2

the R2 component is:

E-PNP-G7 10mmol/L

1.0mmol/L of cerium nitrate

The kit of embodiment 1-2 is suitable for a semi-automatic or full-automatic biochemical analyzer with a thermostat with a corresponding wavelength of 405nm and 37 ℃.

Example 3

First, the reactivity verification test of amylase and substrate can be improved by improving the formula

The lowest detection limit is: the determination of the lowest detection limit uses the same batch number reagent to perform at least 20 times of repeated detection on the zero-concentration calibrator (or sample diluent), and the SD of the average value minus 2 times is the lowest detection limit of the reagent of the average value plus 2 times of SD of the zero-point average value.

1. The reactivity of the amylase in the kits of the two inventions was compared by performing a minimum detection limit validation test on the self-developed alpha-amylase assay kit of the formulation described in example 1 and the alpha-amylase assay kit of the formulation described in patent 201410401270.3. The results are as follows:

2. the reactivity of the amylase in the kits of the two inventions was compared by performing a minimum detection limit validation test on the self-developed alpha-amylase assay kit of the formulation described in example 2 with the alpha-amylase assay kit of the formulation described in patent 201410401270.3. The results are as follows:

in conclusion, the detection limits of the self-prepared examples 1 and 2 are 5.061U/L and 5.105U/L respectively, and the detection limit of the alpha-amylase determination kit with the improved formula disclosed in patent 201410401270.3 is 11.026U/L, so that the improved formula has a better improvement effect on the amylase reactivity than the improved formula disclosed in patent 201410401270.3, and the sensitivity of the detection kit can be further improved, so that the amylase reactivity effect of the amylase determination kit with the improved formula is enhanced, and the sensitivity is further improved. Therefore, the detection method of the kit for measuring alpha-amylase in serum provided by the invention better conforms to the standard of a reference method established by IFCC (enzyme field chromatography), solves the problem of clinical application at present, and has practical clinical application value.

Example 4

Second, test accuracy verification test

1. The kit of the embodiment 1 and the alpha-amylase kit produced by Shanghai Kehua bioengineering GmbH are used for carrying out the correlation verification test of the kit and the alpha-amylase kit by adopting a Beckmann DxC600 full-automatic biochemical analyzer, and the serum of 50 patients is taken for testing and the correlation of the test results of the two is compared.

The results show that the self-developed alpha-amylase assay kit of self-prepared example 1 has good performance consistency with the gold-labeled kit produced by Shanghai Kehua bioengineering GmbH.

2. The kit of the embodiment 2 and the alpha-amylase kit produced by Shanghai Kehua bioengineering GmbH are used for carrying out the correlation verification test of the kit and the alpha-amylase kit by adopting a Beckmann DxC600 full-automatic biochemical analyzer, and the serum of 50 patients is taken for testing and the correlation of the test results of the two is compared.

The results show that the self-developed alpha-amylase assay kit of the self-prepared example 2 has good performance consistency with the gold-labeled kit produced by Shanghai Kehua bioengineering GmbH.

Example 5

Third, the stability of this kit verifies the experiment

1. Stability of the kit of parts of example 1

The alpha-amylase determination kit with the formula in the embodiment 1 is used for stability assessment under the storage condition of 2-8 ℃, and the change situation of the determination value is detected once every three months and reaches 13 months.

2. Stability of the kit of parts of example 2

The alpha-amylase determination kit of the formula in the embodiment 2 is subjected to stability assessment under the storage condition of 2-8 ℃, and the change situation of the determination value is detected once every three months and reaches 13 months.

The results show that the alpha-amylase determination kit has good stability when stored at 2-8 ℃ for 13 months, and the measured value is within the tolerance range of the target value (the deviation between the measured value and the target value should be +/-10%).

By integrating the information, the alpha-amylase determination kit in the self-researched serum can keep good consistency with the detection result of a gold-labeled kit and accurate and reliable detection result on the basis of improving the formula, the kit is stored for 13 months under the storage condition, has stable performance, has better improvement effect on the amylase reactivity, and can further improve the sensitivity of the detection kit, so that the amylase determination kit with the improved formula has the advantages of enhanced amylase reactivity effect and further improved sensitivity. Therefore, the detection method of the kit for measuring alpha-amylase in serum provided by the invention better conforms to the standard of a reference method established by IFCC (enzyme field chromatography), and the problem of current clinical application is solved. Has more practical clinical application value.

It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should also be understood that various alterations, modifications and/or variations can be made to the present invention by those skilled in the art after reading the technical content of the present invention, and all such equivalents fall within the protective scope defined by the claims of the present application.

Claims

1. A kit for measuring alpha-amylase in serum is characterized in that the kit is a liquid double-reagent kit and consists of a reagent 1 and a reagent 2,

the reagent 1 consists of the following components:

the reagent 2 consists of the following components:

0.1-10 mmol/L of 4, 6-ethylene-4-nitrophenol-alpha-heptose;

0.1-1 mmol/L of cerium nitrate.

2. The kit for measuring alpha-amylase in serum according to claim 1, wherein the kit is a liquid dual reagent kit, and is composed of a reagent 1 and a reagent 2,

the reagent 1 consists of the following components:

the reagent 2 consists of the following components:

5mmol/L of 4, 6-ethylene-4-nitrophenol-alpha-heptose;

and 0.5mmol/L of cerium nitrate.