阅读说明：本技术 一种血清中甘氨酰脯氨酸二肽氨基肽酶测定试剂盒 (Kit for determining glycylproline dipeptide aminopeptidase in serum ) 是由 刘阳 孟令洋 夏冬梅 孙成艳 武振宁 于浩滢 张云 于 2020-06-15 设计创作，主要内容包括：一种血清中甘氨酰脯氨酸二肽氨基肽酶测定试剂盒,属于试剂盒技术领域。本发明的试剂盒,包含R1试剂和R2试剂；R1试剂为含有保护剂和防腐剂的缓冲液；缓冲液为3-[(1,1-二甲基-2-羟乙基)氨基]-2-羟基丙磺酸缓冲液、三乙醇胺缓冲液或者三羟基氨基甲烷-盐酸缓冲液；保护剂为双甘肽；防腐剂为Proclin300；R2试剂为含有表面活性剂、防腐剂和底物的缓冲液；缓冲液为酒石酸缓冲液；表面活性剂为烷基酚的聚氧乙烯醚和脂肪醇聚氧乙烯醚中的一种或两种；防腐剂为Proclin300；底物为甘氨酰脯氨酰对硝基苯胺。该试剂盒,试剂开封稳定性和高温稳定性高,重复性好；避免了临床测试的假性结果,检测效率和准确度高。(A kit for determining glycylproline dipeptidyl aminopeptidase in serum belongs to the technical field of kits. The kit of the invention comprises an R1 reagent and an R2 reagent; the R1 reagent is a buffer solution containing a protective agent and a preservative; the buffer solution is 3- [ (1, 1-dimethyl-2-hydroxyethyl) amino ] -2-hydroxypropanesulfonic acid buffer solution, triethanolamine buffer solution or trihydroxyaminomethane-hydrochloric acid buffer solution; the protective agent is diglycine; the preservative is Proclin 300; the R2 reagent is a buffer solution containing a surfactant, a preservative and a substrate; the buffer solution is tartaric acid buffer solution; the surfactant is one or two of polyoxyethylene ether and fatty alcohol polyoxyethylene ether of alkylphenol; the preservative is Proclin 300; the substrate is glycylprolyl p-nitroaniline. The reagent kit has high unsealing stability and high-temperature stability of the reagent, and good repeatability; false results of clinical tests are avoided, and the detection efficiency and accuracy are high.)

1. A kit for determining glycylproline dipeptidyl aminopeptidase in serum is characterized in that,

comprising an R1 reagent and an R2 reagent;

the R1 reagent is a buffer solution containing a protective agent and a preservative; the buffer solution is 3- [ (1, 1-dimethyl-2-hydroxyethyl) amino ] -2-hydroxypropanesulfonic acid buffer solution with the concentration range of 50-100mM, triethanolamine buffer solution with the concentration range of 8-10g/L or trihydroxyaminomethane-hydrochloric acid buffer solution with the concentration range of 60-100 mM; the protective agent is diglycine, and the concentration of the protective agent in the R1 reagent is 5-50 mM; the preservative is Proclin300, and the concentration of the preservative in the R1 reagent is 0.05 wt% -0.25 wt%;

the R2 reagent is a buffer solution containing a surfactant, a preservative and a substrate; the buffer solution is tartaric acid buffer solution with the concentration range of 20-100 mM; the surfactant is one or two of polyoxyethylene ether and fatty alcohol polyoxyethylene ether of alkylphenol, and the concentration of the surfactant in the R2 reagent is 0.1-3 wt%; the preservative is Proclin300, and the concentration of the preservative in the R2 reagent is 0.05 wt% -0.25 wt%; the substrate is glycylprolyl p-nitrobenzene, and the concentration of the substrate in the R2 reagent is 6 mM.

2. The kit for the determination of glycylproline dipeptidylaminopeptidase in serum as claimed in claim 1, wherein the buffer in the reagent R1 is 3- [ (1, 1-dimethyl-2-hydroxyethyl) amino ] -2-hydroxypropanesulfonic acid buffer at a concentration of 80 mM.

3. The kit for assaying glycylproline dipeptidyl aminopeptidase in serum according to claim 1, wherein the concentration of the protective agent in the R1 reagent is 20 mM.

4. The kit for assaying glycylproline dipeptidyl aminopeptidase in serum according to claim 1, wherein the concentration of the preservative in the reagent R1 is 0.1 wt%.

5. The kit for assaying glycylproline dipeptide aminopeptidase in serum according to claim 1, wherein the concentration of the tartaric acid buffer in the reagent R2 is 50 mM.

6. The kit for assaying glycylproline dipeptide aminopeptidase in serum as claimed in claim 1, wherein in the reagent R2, the surfactant is laureth alcohol at a concentration of 2 wt%.

7. The kit for assaying glycylproline dipeptidyl aminopeptidase in serum according to claim 1, wherein the concentration of the preservative in the reagent R2 is 0.1 wt%.

Technical Field

The invention belongs to the technical field of kits, and particularly relates to a kit for determining glycylproline dipeptidyl aminopeptidase in serum.

Background

From commercial enzyme product Acylase I, Hopsu-Havu and Glenner, glycylproline dipeptide aminopeptidase (GPDA) was first discovered and isolated in 1966, and then GPDA was confirmed in tissues such as rat liver, rat kidney, pig kidney, human and various animal sera, human salivary gland and saliva, bovine and human dental pulp and gum.

GPDA is mainly found in the salivary gland, submandibular gland and other tissues of the human body, and saliva and blood also contain the enzyme. The main role of GPDA is to hydrolyze the peptide chain between glycylproline and other amino acids located at the N-terminus of the peptide chain. The enzyme activity in blood serum of liver disease patients is increased, and gastric cancer patients are decreased. The GPDA activity in the serum of patients with primary liver cancer (PHC) and secondary liver cancer is obviously higher than that in patients with chronic hepatitis, liver cirrhosis, cholelithiasis, obstructive jaundice and normal control group. Acute hepatitis, chronic live liver, liver cirrhosis, obstructive jaundice, etc., and GPDA in blood serum may be increased to different extent, which is not as high as that of liver cancer patients. However, in severe hepatitis and alcoholic hepatitis, GPDA in blood serum is higher than that in liver cancer patients. The GPDA of the serum of a patient with gastric cancer is obviously reduced, and is generally about 1/2 of a normal person. Other benign gastrointestinal lesions. The GPDA also decreased slightly. The gastric ulcer, which is the major descending factor, is chronic gastritis and duodenal bulbar ulcer. After gastric cancer resection, GPDA in the serum of a patient tends to rise.

Disclosure of Invention

The invention aims to solve the technical problem of instability of reagents used in the detection method of the glycylproline dipeptide aminopeptidase in the prior art, and provides a kit for determining the glycylproline dipeptide aminopeptidase in serum.

The invention provides a kit for determining glycylproline dipeptide aminopeptidase in serum,

comprising an R1 reagent and an R2 reagent;

the R1 reagent is a buffer solution containing a protective agent and a preservative; the buffer solution is 3- [ (1, 1-dimethyl-2-hydroxyethyl) amino ] -2-hydroxypropanesulfonic Acid (AMPSO) buffer solution with the concentration range of 50-100mM, triethanolamine buffer solution with the concentration range of 8-10g/L or trihydroxyaminomethane-hydrochloric acid (Tris-HCl) buffer solution with the concentration range of 60-100 mM; the protective agent is diglycine, and the concentration of the protective agent in the R1 reagent is 5-50 mM; the preservative is Proclin300, and the concentration of the preservative in the R1 reagent is 0.05 wt% -0.25 wt%;

the R2 reagent is a buffer solution containing a surfactant, a preservative and a substrate; the buffer solution is tartaric acid buffer solution with the concentration range of 20-100 mM; the surfactant is one or two of polyoxyethylene ether and fatty alcohol polyoxyethylene ether of alkylphenol, and the concentration of the surfactant in the R2 reagent is 0.1-3 wt%; the preservative is Proclin300, and the concentration of the preservative in the R2 reagent is 0.05 wt% -0.25 wt%; the substrate is glycylprolyl p-nitrobenzene, and the concentration of the substrate in the R2 reagent is 6 mM.

Preferably, in the R1 reagent, the buffer is 3- [ (1, 1-dimethyl-2-hydroxyethyl) amino ] -2-hydroxypropanesulfonic acid buffer with a concentration of 80 mM.

Preferably, the concentration of the protective agent in the R1 reagent is 20 mM.

Preferably, the concentration of the preservative in the R1 reagent is 0.1 wt%.

Preferably, the concentration of the tartaric acid buffer in the R2 reagent is 50 mM.

Preferably, in the R2 reagent, the surfactant is polyoxyethylene lauryl ether, and the concentration is 2 wt%.

Preferably, the concentration of the preservative in the R2 reagent is 0.1 wt%.

Compared with the prior art, the invention has the beneficial effects that:

according to the kit for determining glycylproline dipeptide aminopeptidase in serum, tartaric acid is used, so that the unsealing stability and the high-temperature stability of a reagent are improved, and the detection repeatability is good; the method effectively avoids the false result of clinical test, greatly improves the detection efficiency and accuracy, is suitable for various full-automatic biochemical analyzers, has wider universality and is convenient to popularize.

The kit for determining glycylproline dipeptidyl aminopeptidase in serum has lower raw material cost, can replace imported reagents, and saves the expense of patients.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the detailed description will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a calibration curve of the kit for assaying glycylproline dipeptidyl aminopeptidase in serum provided in example 1 of the present invention.

FIG. 2 is a method of using the kit for glycylproline dipeptidyl aminopeptidase in serum according to the present invention.

Detailed Description

For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the detailed description, but it is to be understood that the description is intended to further illustrate the features and advantages of the invention and not to limit the claims to the invention.

The kit for determining glycylproline dipeptidyl aminopeptidase in serum comprises an R1 reagent and an R2 reagent.

Wherein, the R1 reagent is a buffer solution containing a protective agent and a preservative; the buffer solution is 3- [ (1, 1-dimethyl-2-hydroxyethyl) amino ] -2-hydroxypropanesulfonic acid buffer solution with the concentration range of 50-100mM, triethanolamine buffer solution with the concentration range of 8-10g/L or trihydroxyaminomethane-hydrochloric acid buffer solution with the concentration range of 60-100 mM; the protective agent is diglycine, and the concentration of the protective agent in the R1 reagent is 5-50 mM; the preservative is Proclin300, and the concentration of the preservative in the R1 reagent is 0.05 wt% -0.25 wt%;

the R2 reagent is a buffer solution containing a surfactant, a preservative and a substrate; the buffer solution is tartaric acid buffer solution with the concentration range of 20-100 mM; the surfactant is one or two of polyoxyethylene ether and fatty alcohol polyoxyethylene ether of alkylphenol, and the concentration of the surfactant in the R2 reagent is 0.1-3 wt%; the preservative is Proclin300, and the concentration of the preservative in the R2 reagent is 0.05 wt% -0.25 wt%; the substrate is glycylprolyl p-nitrobenzene, and the concentration of the substrate in the R2 reagent is 6 mM.

In the above technical means, the buffer solution in the R1 reagent is preferably 3- [ (1, 1-dimethyl-2-hydroxyethyl) amino ] -2-hydroxypropanesulfonic acid buffer solution with a concentration of 80 mM.

In the above embodiment, the concentration of the protecting agent in the R1 reagent is preferably 20 mM.

In the above embodiment, the concentration of the preservative in the R1 reagent is preferably 0.1 wt%.

In the above embodiment, the concentration of the tartaric acid buffer in the R2 reagent is preferably 50 mM.

In the technical scheme, in the R2 reagent, the surfactant is preferably polyoxyethylene lauryl ether, and the concentration is preferably 2 wt%.

In the above embodiment, the concentration of the preservative in the R2 reagent is preferably 0.1 wt%.

The detection principle of the kit for determining glycylproline dipeptidyl aminopeptidase in serum provided by the invention is as follows: under alkaline conditions, GPDA catalyzes the substrate glycylprolyl-p-nitroaniline to be hydrolyzed to generate glycylproline and yellow p-nitroaniline, the latter can cause the increase of absorbance at specific wavelength, and the increase rate of the absorbance is in direct proportion to the activity of GPDA.

The method of using the kit for assaying glycylproline dipeptidyl aminopeptidase in serum according to the present invention is shown in FIG. 2 and Table 1.

TABLE 1

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the following embodiments and the accompanying drawings.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.