阅读说明：本技术 一种分析灵敏度高的甘氨酰脯氨酸二肽氨基肽酶测定试剂盒及其制备方法和应用 (Glycylproline dipeptide aminopeptidase determination kit with high analysis sensitivity and preparation method and application thereof ) 是由 刘安娜 于 2020-03-20 设计创作，主要内容包括：本发明公开了一种甘氨酰脯氨酸二肽氨基肽酶测定试剂盒,试剂盒含有试剂R1和试剂R2,试剂R1中含有以下成分：缓冲液、双甘氨肽、羧甲基壳聚糖、氯化钠、防腐剂；试剂R2中含有以下成分：缓冲液、甘氨酰-脯氨酰-对硝基苯胺对甲苯磺酸盐、稳定剂、表面活性剂、防腐剂。本发明同时提供了该试剂盒的制备方法和应用,该试剂盒是一种灵敏度高、稳定性强、重复性好的液体试剂盒。(The invention discloses a glycylproline dipeptide aminopeptidase measuring kit, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components: buffer solution, glycylglycine, carboxymethyl chitosan, sodium chloride and preservative; the reagent R2 contains the following components: buffer solution, glycyl-prolyl-p-nitroaniline p-toluenesulfonate, a stabilizer, a surfactant and a preservative. The invention also provides a preparation method and application of the kit, and the kit is a liquid kit with high sensitivity, strong stability and good repeatability.)

1. A glycylproline dipeptide aminopeptidase assay kit, which is characterized in that the kit contains a reagent R1 and a reagent R2;

the reagent R1 contains the following components:

buffer 50-200 mmol/L

Glycylglycine 20-120 mmol/L

Carboxymethyl chitosan 1-20 g/L

Sodium chloride 1-20 g/L

0.5-2 g/L g of preservative

The reagent R2 contains the following components:

the buffer solution is 50-200 mmol/L,

glycyl-prolyl-p-nitroaniline p-toluenesulfonate 10-100 mmol/L

Stabilizer 1-20 g/L

Surfactant 1-20 g/L

0.5-2 g/L of preservative.

2. The glycylproline dipeptide aminopeptidase assay kit according to claim 1, wherein the buffer in reagent R1 is a 2-amino-2-methyl-1-propanol buffer; the buffer solution in the reagent R2 is N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid buffer solution.

3. The glycylproline dipeptide aminopeptidase assay kit according to claim 1 wherein the pH of reagent R1 is 7.0-10.0; the pH value of the reagent R2 is 6.0-10.0.

4. The glycylproline dipeptide aminopeptidase assay kit of claim 1 wherein the stabilizing agents in reagent R2 are sodium alginate and dextran sulfate.

5. The glycylproline dipeptide aminopeptidase assay kit according to claim 1, wherein the preservative in reagent R1 is one or more of sodium azide, PC300, imidazolidinyl urea, methylparaben; the preservative in the reagent R2 is one or more of sodium azide, PC300, imidazolidinyl urea and methyl p-hydroxybenzoate.

6. The glycylproline dipeptide aminopeptidase measurement kit according to claim 1, wherein the surfactant in the reagent R2 is one or two of polyvinylpyrrolidone k12 and alkylphenol ethoxylate op-10.

7. The glycylproline dipeptide aminopeptidase measurement kit according to claim 1, wherein the volume ratio of the reagent R1 to the reagent R2 is 1-5: 1.

8. The glycylproline dipeptide aminopeptidase assay kit of claim 1 wherein the volume ratio of reagent R1 to reagent R2 is 3: 1.

9. A method for preparing a glycylproline dipeptide aminopeptidase assay kit according to any of claims 1 to 8 comprising the steps of:

(1) preparation of reagent R1: taking a proper amount of water, respectively and sequentially adding the raw materials shown in R1, stirring uniformly to dissolve one raw material, adding the next raw material, adjusting the pH value to 7.0-10.0 by using hydrochloric acid or sodium hydroxide, and fixing the volume to the required volume;

(2) preparation of reagent R2: adding appropriate amount of water into R2, stirring to dissolve one material, adding the next material, adjusting pH to 6.0-10.0 with hydrochloric acid or sodium hydroxide, and diluting to desired volume.

10. Use of a kit according to any one of claims 1 to 9 for determining the concentration of glycylproline dipeptidyl aminopeptidase in serum for non-disease diagnostic and therapeutic purposes.

Technical Field

The invention relates to the technical field of biochemical reagent determination, in particular to a glycylproline dipeptide aminopeptidase determination kit with high analysis sensitivity and a preparation method and application thereof.

Background

Glycylproline dipeptidyl aminopeptidase (GPDA) is first discovered in 1966, widely distributed in liver, kidney, connective tissue, salivary gland, serum, saliva and other body fluids, and most (55%) of GPDA in cells is distributed in microsomes; GPDA in the soluble fraction of the cell, the nucleus and the mitochondria accounted for 20%, 18% and 8% of the total intracellular GPDA activity, respectively. The glycylproline dipeptide aminopeptidase can specifically hydrolyze and release glycyl-proline (Gly-Pro) at the N-terminal of a peptide chain, the collagen molecule is rich in the Gly-Pro structure, and the physiological action of GPDA can be related to collagen peptide degradation. Collagen is a protein having the highest content in the body, is a main component of connective tissue, is also a main component of epithelial cell basement membrane, and has an important role in maintaining the functions of these tissues, and cancer tissues generated by differentiation of epithelial cells or mesenchymal cells often contain collagenase, which decomposes collagen around cancer tissues, may be involved in infiltration and diffusion of cancer, and GPDA may also play an important role as a dipeptidylaminopeptidase in the collagen decomposition process.

Studies have shown that the GPDA levels of healthy adult serum are relatively constant, with neonates significantly lower than adults. From the stage of growth and development when the infant is born to the age of 20 years, the serum GPDA rises gradually. This may be associated with an increase in collagen amounts, also suggesting a role for GPDA in collagen peptide degradation. The GPDA activity of the serum of the primary liver cancer patient is increased and is obviously higher than that of the patients with liver cirrhosis, cholelithiasis and chronic hepatitis, the determination of the GPDA activity has certain specificity to the diagnosis of the primary liver cancer and possibly reflects the hyperfunction of collagen catabolism of the primary liver cancer patients. The activity of the glycylproline dipeptide aminopeptidase in the serum of the gastric cancer patient is obviously reduced, and the activity of the glycylproline dipeptide aminopeptidase in the serum is slightly reduced when other benign gastrointestinal diseases exist. GPDA in serum has two isoenzymes, GPDA-S and GPDA-F, GPDA-F is an important marker of primary liver cancer when the content of AFP is not obviously changed, and has good complementary action with AFP (Run-Zhou Ni et al 2003).

The glycylproline dipeptide aminopeptidase test method comprises a fluorescence method, an enzyme-linked immunosorbent assay and a colorimetric method. The fluorescence method and the enzyme-linked immunosorbent assay have high sensitivity, but the operation is relatively complex, and the fluorescence method is generally used for measuring urine samples with low GPDA level. The colorimetric method generally adopts a continuous monitoring method, namely GPDA catalyzes a substrate glycylprolyl-p-nitroaniline to be hydrolyzed to generate glycylproline and yellow p-nitroaniline, the latter can cause the increase of absorbance under a specified wavelength, the increase rate of the absorbance is in direct proportion to the activity of the GPDA, and the glycylproline dipeptidyl aminopeptidase measuring reagent in the market has the problems of low analysis sensitivity and poor repeatability, and the problems are particularly obvious in instruments and equipment with poor precision. The kit for detecting glycylproline dipeptide aminopeptidase by using a continuous monitoring method is provided by repeatedly testing and optimizing a reaction system, has good stability, high sensitivity and good repeatability, and can be used for batch detection of clinical samples.

Disclosure of Invention

In order to solve the problems, the invention provides a glycylproline dipeptide aminopeptidase measuring kit, a preparation method and application thereof.

The invention is realized by the following technical scheme: a glycylproline dipeptide aminopeptidase kit comprising a reagent R1 and a reagent R2;

the reagent R1 contains the following components:

the reagent R2 contains the following components:

preferably, the buffer in the reagent R1 is 2-amino-2-methyl-1-propanol (AMP) buffer; the buffer solution in the reagent R2 is N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (Taps) buffer solution.

Preferably, the pH value of the reagent R1 is 7.0-10.0; the pH value of the reagent R2 is 6.0-10.0.

Preferably, the stabilizing agents described in the reagent R2 are sodium alginate and dextran sulfate.

Preferably, the preservative in the reagent R1 is one or more of sodium azide, PC300, imidazolidinyl urea and methyl p-hydroxybenzoate; the preservative in the reagent R2 is one or more of sodium azide, PC300, imidazolidinyl urea and methyl p-hydroxybenzoate.

Preferably, the surfactant in the reagent R2 is one or two of polyvinylpyrrolidone k12 and alkylphenol polyoxyethylene op-10.

Preferably, the volume ratio of the reagent R1 to the reagent R2 is 1-5: 1. More preferably, the volume ratio of reagent R1 to reagent R2 is 3: 1.

The preparation method of the glycylproline dipeptide aminopeptidase measuring kit comprises the following steps:

(1) preparation of reagent R1: taking a proper amount of water, respectively and sequentially adding the raw materials shown in R1, stirring uniformly to dissolve one raw material, adding the next raw material, adjusting the pH value to 7.0-10.0 by using hydrochloric acid or sodium hydroxide, and fixing the volume to the required volume;

(2) preparation of reagent R2: adding appropriate amount of water into R2, stirring to dissolve one raw material, adding the next raw material, adjusting pH to 6.0-10.0 with hydrochloric acid or sodium hydroxide, and diluting to desired volume.

The invention also discloses the application of the kit, which is used for determining the concentration of glycylproline dipeptide aminopeptidase in serum for the purposes of non-disease diagnosis and treatment.

The basic principle of the invention is as follows:

under alkaline conditions, GPDA catalyzes a substrate glycyl-prolyl-p-nitroaniline p-toluenesulfonate to hydrolyze, and glycyl proline and yellow p-nitroaniline can be generated, the latter can cause the increase of absorbance at the wavelength of 405nm, and the increase rate of the absorbance is in direct proportion to the activity of GPDA.

Glycyl-prolyl-p-nitroaniline p-toluenesulfonateGlycylproline + p-nitroaniline

The invention has the beneficial effects that:

1) according to the invention, glycyl-prolyl-p-nitroaniline p-toluenesulfonate is selected as a reaction substrate, the safety of the substrate is high, and simultaneously, the carboxymethyl chitosan is added, so that the synergistic effect on the reaction is achieved, the color development capability after the reaction is strong, and the analysis sensitivity of the reagent is improved.

2) Buffer solutions are added into the reagents R1 and R2, 2-amino-2-methyl-1-propanol (AMP) buffer solution is preferable in the reagent R1, TAPS buffer solution is preferable in the reagent R2, and the buffer solution in the reagent R1 is used for incubation to remove interference, so that the buffer solution in the reagent R2 can effectively improve GPDA catalytic activity, ensure color development effect, inhibit other side reactions, and contribute to improving the repeatability of the reagents.

3) Sodium alginate and dextran sulfate are added into R2 of the reagent, which can synergistically improve the solubility and stability of the substrate, and simultaneously, the surfactant is added, which has emulsification and dissolution assistance, can further improve the stability of the substrate solution, and stimulates GPDA enzyme to exert maximum activity.

Drawings

FIG. 1 is a correlation curve for the reagents of example 1 and comparative example 1;

FIG. 2 is a correlation curve for the reagents of comparative example 2 and comparative example 1;

FIG. 3 is a graph showing the change in concentration of the glycylproline dipeptide aminopeptidase measuring reagent for stability test provided in example 1 and comparative examples 1, 2, 4, 5, 6 and 7.

Detailed Description

The following is a description of specific embodiments of the present invention with reference to the drawings, and the technical solutions of the present invention will be further described, but the present invention is not limited to these embodiments.

The test conditions of the kit for determining glycylproline dipeptide aminopeptidase in serum are as follows: the method comprises the following steps: a rate method; primary/secondary wavelength: 405nm/800 nm; temperature: 37 ℃; the correction type is as follows: linearity; the calibration method comprises the following steps: calibrating at two points; the reaction direction is as follows: upwards.

The specific operation is shown in table 1.

TABLE 1 glycylproline dipeptidyl aminopeptidase assay reagent protocols

And (3) calculating the result:

sample requirements:

1. insoluble blood serum.

2. Sample stability: the specimen can be stored stably for 3 days at the temperature of 2-8 ℃ and for 2 weeks at the temperature of-20 ℃.