阅读说明：本技术 一种1,5-脱水山梨醇检测试剂盒及其制备方法 (1, 5-sorbitan detection kit and preparation method thereof ) 是由 袁嘉扬 张凤 于 2019-11-01 设计创作，主要内容包括：本发明涉及一种1,5-脱水山梨醇检测试剂盒,包括试剂R1和试剂R2,试剂R1的各组分及浓度包括：20-100mmol/L的第一缓冲液、1-20KU/L的葡萄糖激酶、0.5-5KU/L的丙酮酸激酶、0.2-1mmol/L的三磷酸腺苷、1-3mmol/L的磷酸烯醇式丙酮酸钾、0.5-2mmol/L的4-氨基安替比林、1-5ml/L的表面活性剂、0.5—10g/L的谷胱甘肽、0.8-2.0ml/L的防腐剂、5-20mmol/L的氯化镁。试剂R2的各组分及浓度包括：20-100mmol/L的第二缓冲液、20-125KU/L的吡喃糖氧化酶、15-60KU/L的过氧化物酶、1-10mmol的色原、1-5ml/L的表面活性剂、0.8-2.0ml/L的防腐剂。本发明的优点是：灵敏度高、稳定性好、线性范围宽。(The invention relates to a 1, 5-sorbitan detection kit, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in percentage by weight: 20-100mmol/L of first buffer solution, 1-20KU/L of glucokinase, 0.5-5KU/L of pyruvate kinase, 0.2-1mmol/L of adenosine triphosphate, 1-3mmol/L of potassium phosphoenolpyruvate, 0.5-2mmol/L of 4-aminoantipyrine, 1-5ml/L of surfactant, 0.5-10 g/L of glutathione, 0.8-2.0ml/L of preservative, and 5-20mmol/L of magnesium chloride. The components and concentrations of the reagent R2 include: 20-100mmol/L of second buffer solution, 20-125KU/L of pyranose oxidase, 15-60KU/L of peroxidase, 1-10mmol of chromogen, 1-5ml/L of surfactant and 0.8-2.0ml/L of preservative. The invention has the advantages that: high sensitivity, good stability and wide linear range.)

1. The utility model provides a 1, 5-sorbitan assay kit which characterized in that: the reagent comprises a reagent R1 and a reagent R2, wherein each component and concentration of the reagent R1 comprises:

the reagent R2 comprises the following components in percentage by weight:

2. the 1, 5-sorbitan assay kit according to claim 1, wherein: the first buffer solution in the reagent R1 and the second buffer solution in the reagent R2 are respectively one or a mixture of more than two of potassium phosphate buffer solution, HEPES buffer solution, MES buffer solution and Tris buffer solution.

3. The 1, 5-sorbitan assay kit according to claim 1, wherein: the preservatives in the reagents R1 and R2 are respectively one or a mixture of more than two of sodium azide, Proclin950, Proclin300 and thimerosal.

4. The 1, 5-sorbitan assay kit according to claim 1 or 2, wherein: the surfactant in the reagents R1 and R2 is one or a mixture of more than two of Tween 20, Triton X-100, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether.

5. The 1, 5-sorbitan assay kit according to claim 4, wherein: the chromogen in the reagent R2 is one of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt and 3-hydroxy-2, 4, 6-triiodobenzoic acid (HTIB).

6. The method for producing a 1, 5-sorbitan test kit according to any one of claims 1 to 5, characterized in that: comprises the following steps of (a) carrying out,

(1) preparation of reagent R1

Adding part of purified water into a liquid preparation tank, adding a stirrer to a magnetic stirrer, regulating the rotating speed of the stirrer to 200-300rpm, keeping stirring in a constant speed state, adding a first buffer solution while stirring according to the concentration requirement of claim 1, stirring until the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, then adding glucokinase, pyruvate kinase, adenosine triphosphate, phosphoenolpyruvate potassium pyruvate and 4-aminoantipyrine, sequentially adding glutathione, magnesium chloride, a surfactant and an antiseptic according to the feeding mode after the materials are completely dissolved, continuing stirring until all the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, adjusting the pH value to be 6.50-8.00, and fixing the volume to the final volume;

filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;

(2) preparation of reagent R2

Adding part of purified water into a liquid preparation tank, adding a second buffer solution while stirring according to the concentration requirement of claim 1, stirring until the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, adding pyranose oxidase and peroxidase, sequentially adding the chromogen, the surfactant and the preservative according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, adjusting the pH value and fixing the volume to the final volume.

And filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution in a finished product tank for marking.

Technical Field

The invention relates to the technical field of biology, in particular to a 1, 5-sorbitan kit and a preparation method thereof.

Background

1,5-AG (1, 5-anhydro-sorbitol) shows obvious negative correlation with the existing diabetes index, and the degree of reduction of 1,5-AG in blood is obviously correlated with the severity of diabetes. 1,5-AG reflects the average blood glucose level of several days to 1 week, and thus the effect of 1, 5-sorbitan detection is increasingly emphasized.

The existing reagent and the existing detection method for detecting the 1, 5-anhydro-sorbitol have the defects of poor sensitivity and repeatability and low accuracy, and limit the application of the reagent and the detection method. Therefore, a new technical solution should be provided to solve the above problems.

Disclosure of Invention

The purpose of the invention is: provides a 1, 5-dehydrated sorbitol detection kit which can obviously improve the repeatability and the sensitivity and a preparation method thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

the 1, 5-sorbitan detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following components in concentration:

the reagent R2 comprises the following components in percentage by weight:

the further technical scheme is as follows:

the first buffer solution in the reagent R1 and the second buffer solution in the reagent R2 are respectively one or a mixture of more than two of potassium phosphate buffer solution, HEPES buffer solution, MES buffer solution and Tris buffer solution.

The preservatives in the reagents R1 and R2 are respectively one or a mixture of more than two of sodium azide, Proclin950, Proclin300 and thimerosal.

The surfactant in the reagents R1 and R2 is one or a mixture of more than two of Tween 20, Triton X-100, polyvinylpyrrolidone and octyl phenyl polyoxyethylene ether.

The chromogen in the reagent R2 is one of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt and 3-hydroxy-2, 4, 6-triiodobenzoic acid.

A method for preparing a 1, 5-dehydrated sorbitol detection kit comprises the following steps,

(1) preparation of reagent R1

Adding part of purified water into a liquid preparation tank, adding a stirrer to a magnetic stirrer, adjusting the rotating speed of the stirrer to 200 plus 300rpm, keeping stirring in a uniform speed state, adding a first buffer solution while stirring according to the concentration requirement, stirring until the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, then adding glucokinase, pyruvate kinase, adenosine triphosphate, phosphoenolpyruvate potassium pyruvate and 4-aminoantipyrine, sequentially adding glutathione, magnesium chloride, a surfactant and an antiseptic according to the feeding mode after the materials are completely dissolved, continuing stirring until all the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank after the materials are completely dissolved, adjusting the pH value to be between 6.50 and 8.00, and fixing the volume to the final volume;

filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;

(2) preparation of reagent R2

Adding part of purified water into a liquid preparation tank, adding a second buffer solution while stirring according to the concentration requirement, stirring until the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, adding pyranose oxidase and peroxidase, sequentially adding chromogen, surfactant and preservative according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved and no precipitate is formed at the bottom of the liquid preparation tank, adjusting the pH value and fixing the volume to the final volume.

And filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution in a finished product tank for marking.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

1. according to the 1, 5-sorbitan detection kit, magnesium chloride, glutathione and triton X-100 are simultaneously added into an R1 reagent, so that the activity and interference removal performance of enzyme can be effectively improved, the analysis sensitivity and stability of the reagent are improved, and the accuracy of a detection result is ensured.

2. The invention has the advantages of simple operation, rapid detection, high sensitivity, good stability and wider linear range, and is suitable for being popularized and used in various large, medium and small hospitals.

Drawings

FIG. 1 calibration graph of a calibrator.

Wherein: the X-axis represents calibrator concentration and the Y-axis represents absorbance.

Detailed Description

The invention is further described below with reference to specific examples: