RU1776692C - Reagent for determining concentration of glucose in blood serum - Google Patents

Abstract

Usage: in medical biochemistry and analytical chemistry for the determination of glucose in blood serum. Purpose: improving the accuracy of determination, reducing the cost of the reagent and increasing its stability. The inventive reagent for determining glucose is a mixture of the following ingredients: glucose oxidase 4,5-9,0 PIECES, peroxidase 0.25-2.0 PIECES, 4-aminoantipyrine 0,019-0,056 mg, phenol 0.27-1.29-mg , horse serum albumin 5-10 mg, phosphate buffered saline pH 6.2-7.2 - 1 ml. Benefit: reagent consumption is reduced, repeatability of the assay is increased, and the shelf life of the reagent is increased to 7 weeks. next to

Description

The invention relates to medical biochemistry, analytical chemistry, and in particular to the creation of reagent kits that allow the determination of glucose in blood serum by an enzymatic method.

The kits are based on the enzymatic method, in which glucose is oxidized by atmospheric oxygen by the catalytic action of glucose oxidase (GOD), with the formation of hydrogen peroxide and / -gluconolactone. The resulting hydrogen peroxide is determined by the i-indophenol reaction of 4-aminoantipyrine (4-AAA) with a peroxidase-catalyzed chromogen. The amount of dye formed at

compliance with the operating conditions is proportional to the glucose content.

Reagent kits are known for the determination of serum glucose by an enzymatic method.

A known set of reagents for the determination of glucose by the enzymatic method, comprising the following components in the indicated concentrations:

1. A mixture of enzymes

Glucose oxidase

Peroxidase

4-aminoantipyrine

-0.22 g

-4 uct./ml

-1 unit act./ml - 0.34 mmol / l

V4 Os Oh Yu N3

Sodium sulfate up to 0.22 g

2. Chromogen (1.0 g) - 4-chloro-3-methylphenol 0.3 mmol / L with Slovasol 0 and sodium sulfate.

3. The buffer solution is Tris 0.91 mol / L pH 8.3.

4. The standard of glucose is 25 mmol / l. The disadvantages of this kit are:

1. Short shelf life of the reagent kit in solution, which is 1-2 weeks.

2. The use of inaccessible reagents as chromogen - 4-chloro-3-methylphenol and Slovasol 0.

3. In the practical laboratory, the reproducibility of the method using this kit is small and amounts to 8.8%.

Closest to the claimed is a set of reagents for the determination of glucose by the enzymatic method, which consists of the following components in the indicated concentrations:

1. Buffer, enzymes ;, 4-AAP - phosphate buffer 100 mmol / l pH 7.0; YEAR 18 units.act./ml; peroxidase 1.1 unit / ml; 4-AAP - 0.77 mmol / L

2. Phenol - 11 mmol / L

3. Ethanol glucose - 0.505; 5.55 mmol / L.

The disadvantages of the prototype are:

1. High reagent consumption: YEAR- 18 units.act./ml; phenol - 11 mmol / l; 4-AAL - 0.77 mmol / L.

2. Short shelf life of the kit in solution: 4 weeks at a temperature of (2-8) ° C.

3. The difficulty in preparing the kit due to the fixed concentration values of the components of the kit.

The aim of the invention is to improve the reproducibility of the assay, reduce the cost of the reagent and increase its stability while maintaining the correct determination of glucose.

The goal is achieved by creating a kit containing the following components in the indicated concentrations:

Enzyme mixture: with the ratio of activities YEAR / peroxidase from 18 to 4.5

1.YEAR - 4.5-9.0 units / ml

2. Peroxidase - 0.25-2.0 units.act./ml

3.4-AAP - 0.19-0.056 mg / ml

4.Phenol - 0.27-1.29 mg / ml

5. Horse serum albumin (LSA) -0.1-10.0 mg / ml

6. The buffer solution is 0.1 mol / l, potassium phosphate (pH 7.0 ± 0.5) - the rest.

The hallmarks of the reagent kit are:

- the selected ratio of the concentrations of the components of the kit;

- the introduction of the stabilizer LSA into the enzyme mixture, which allowed:

1. To reduce the consumption of reagents, due to which significantly reduce the cost of the cost of 5 boron.

2. To increase the reproducibility of the analysis (with LSA - 3.9%, without LSA - 5.7%).

3. Increase the shelf life of the kit solution to 7 weeks.

0 The invention is illustrated by examples.

Enzyme activity was determined by known methods.

Example 1. To 1 ml of kit solution

Five reagents (the concentration of which is indicated in the table) added 10 µl Preclllp serum (its glucose content was 5.67-6.53 mmol / L). The solution was stirred, kept 40 min at room

0 temperature and measured the optical density of the test solution and glucose standard with a concentration of 10 mmol / L on a spectrophotometer at a wavelength of 510 nm in quartz cuvettes with a light-absorbing layer 1 cm thick. The analysis results are given in Table. 1.

As can be seen from the table. 1, when the ratio of YEAR / peroxidase activities is from 4.5 to 18, glucose is determined by this set of glucose

0 is correct (No. 1 - 3.6). With a larger - 22.5 and a smaller - 3.0 ratio of YEAR / peroxidase activities, the analysis results are incorrect (No. 4 - 5).

Example 2. The analysis is analogous to example 1. The concentration of the components and the results of the analysis are presented in table. 2.

As can be seen from the table. 2, kits correctly detect glucose at a concentration of YEAR

0 from 4.5 to 9 most active./ml (No. 1 -3.5); at a lower concentration of YEAR, glucose is not detected correctly (No. 4).

At a fixed value of YEAR and peroxidase, the sets are correctly determined

5 glucose at a 4-AAP concentration of from 0.019 to 0.056 mg / ml (No. 3.6, 7), at a 4-AAP concentration of less than 0.019 mg / ml and more than 0.056 mg / ml, glucose is not detected correctly (Me 8, 9).

0 Example Z. The analysis is carried out analogously to example 1, the concentration of the components and the analysis results are presented in table. 3.

As can be seen from the table. 3, sets correctly

5, glucose is determined at a phenol concentration of 0.27 to 1.29 mg / ml (No. 1 to 3), and at LSA concentrations of 0.1 to 10 mg / ml (No. 6 to 12). With a lower phenol content - 0.20 mg / ml (No. 4) and a higher phenol content - 1.42 mg / ml (No. 5) and LSA - 11

mg / ml (No. 13) glucose is not detected correctly.

LSD at a concentration of 11 mg / ml forms opalescence in the kit solution and thereby distorts the results of glucose determination. Although an LSA concentration of 0.08 mg / ml allows the glucose content to be correctly determined, it does not provide good reproducibility of the results and does not significantly increase the shelf life of the kit solution.

Claims (1)

The claims A reagent for determining the concentration of glucose in blood serum containing glucose oxidase, peroxidase, 4-aminoantipyrine, phenol and phosphate buffered saline pH 6.8-7.2, characterized in that 0 5 that, in order to increase the accuracy of determination, reduce the cost of the reagent and increase its stability, it additionally contains horse serum albumin in the following ratio of components: Glucose oxidase Peroxidase 4-aminoanthylirin phenol Horse serum albumin Phosphate bu4, 5-9.0 IU 0.25-2.0 IU 0.019-0.056 mg 0.27-1.29 mg 5-10 mg ferric solution pH 6.8-7.2 1 ml In 1 ml of 0.1 mol / L phosphate buffer pH 7.0 + 0.5 solution: YEAR, 5 units.act./ml peroxidase 0.25 units / ml; 4-AAA 0.019 mg / ml; phenol 0.2 mg / ml; LSA 0.1 mg / ml. In 1 ml of 0.1 mol / L phosphate buffered saline pH 7.0 + 0.5: YEAR 9 units / ml; peroxidase 1 unit / ml; 4-AAA 0.056 mg / ml; phenol 1.29 mg / ml; LSA 0.1 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer pH 7.0 + 0.5 solution: YEAR 8 units / ml; peroxidase 1.8 units / ml; 4-AAA 0.056 mg / ml; phenol 0.27 mg / ml; LSA 0.1 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer pH 7.010.5: YEAR 9 units / ml; peroxidase 3 units / ml; A-AAP 0.056 mg / ml; phenol 0.27 mg / ml; LSA 0.1 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer a solution of pH 7.0 ± 0.5: YEAR, 5 units of act / ml; peroxidase 0.2 units / ml; A-AAP 0.056 mg / ml; phenol 0.27 mg / ml; LSA 0.1 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer a solution of pH 7,, 5: YEAR 9 units of act / ml; peroxidase 2 units / ml; AAA 0.056 mg / ml; phenol 0.27 mg / ml; LSA 0.1 mg / ml. 6.30 5.92 6.03 5.5 5.62 6.25 N Name and concentration of components In 1 ml of 0.1 mol / L phosphate buffer pH 7,, 5: YEAR 4.5 units / ml peroxidase 1 unit / ml; 4-DPP 0.056 m phenol 0.2 mg / ml; LSD 0.2 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer a solution of pH 7,, 5: YEAR 9 unit / ml; peroxidase 1.0 unit / ml; 4-AAA 0.056 mg / ml; phenol 0.27 mg / ml; LSD 0.2 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer a solution of pH 7,, 5: YEAR 7 unit / ml; peroxidase 1 unit / ml; 4-APP 0.056 mg / ml; phenol 0.27 mg / ml; LSD 0.2 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer a solution of pH 7, OiO, 5: rOfl 3 units.act./ml; peroxidase 1 unit / ml; 4-AAA 0.056 mg / ml; phenol 0.27 mg / ml; LSA 0.2 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer pH 7.0 ± 0.5: YEAR 18 units / ml; peroxidase 1 unit / ml; 4-AAA 0.056 mg / ml; phenol 0.27 mg / ml; LSA 0.2 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer a solution of pH 7, OiO, 5: rOfl 9 units / ml; peroxidase 1 unit / ml; 4-APP 0.019 mg / ml; phenol 0.27 mg / ml; LSA 0.2 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer pH 7.010.5 solution: YEAR 9 unit / Nl; peroxidase 1 unit / ml; 4-AAA 0.056 mg / ml; phenol 0.2 mg / ml; LSA 0.2 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer a solution of pH 7, OtO, 5: rOfl 9 units / ml; peroxidase 1 unit / ml; 4-APP 0.016 mg / ml; phenol Q, 27 mg / ml; LSA 0.2 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer pH 7.0 ± 0.5: YEAR 9 units / ml; peroxidase 1 unit / ml; 4-AAA 0.062 mg / ml; phenol 0.27 mg / ml; LSA 0.2 mg / ml. table 2 Determined by glucose, mnol / l In 1 ml of 0.1 mol / L phosphate buffer a solution of pH 7,, 5: YEAR E unit / ml; peroxidase 1 unit / ml; 4-AAA 0.050 mg / ml; phenol 0.27 mg / ml; LSA O, 1 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer pH 7.010.5 solution: YEAR $ units / ml; peroxidase 1 unit / ml; 4-AAA 0.050 mg / ml; phenol 1.29 mg / ml; LSA 0.1 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer a solution of pH 7,, 5: YEAR 9 unit / ml; peroxidase 1 unit / ml; 4-AAA 0.050 mg / ml; phenol 0.65 mg / ml; LSA 0.1 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer pH 7.0 ± 0.5: YEAR 9 units / ml; peroxidase 1 unit / ml; 4-AAA 0.050 mg / ml; phenol 0.20 mg / ml; LSA 0.1 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer pH 7.0 ± 0.5: YEAR 9 units / ml; peroxidase 1 unit / ml; 4-AAA 0.050 mg / ml; phenol 1.42 mg / ml; LSA 0.1 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer pH 7.0 ± 0.5: YEAR 9 units / ml; peroxidase 1 unit / ml; 4-APP 0.050 mg / ml; phenol 0.65 mg / ml; LSA 0.2 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer pH 7.0 ± 0.5: YEAR 9 units / ml; peroxidase 1 unit actml; 4-AAA 0.050 mg / ml; phenol 0.65 mg / ml; LSA 0.1 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer pH 7.0 ± 0.5: YEAR 9 units / ml; peroxidase 1 unit / ml; A-AAP 0.050 mg / ml; phenol 0.65 mg / ml; LSA 0.15 mg / ml. In 1 ml of 0.1 mol / L phosphate buffer a solution of pH 7.0 ± 0.5: YEAR 9 units, act./ml; peroxidase 1 unit / ml; -AAA 0.050 mg / ml; phenol 0.65 mg / ml; LSA 0.08 mg / ml. Table3 Continuation of the table. 3 LZZZZZZZZIZ 0 V 1 ml 0.1 mol / L phosphate buffer 5.99 a solution of pH 7,, 5: YEAR 9 unit / ml; peroxidase 1 unit / ml; -AAA 0.050 mg / ml; phenol 0.65 mg / ml; LSA mg / ml. 1B 1 ml of 0.1 mol / L phosphate buffer 5.75 pH 7.0 + 0.5 solution: YEAR 9 units / ml; peroxidase 1 unit / ml; } -AARP 0,050 mg / ml; phenol 0.65 mg / ml; LSA 2 mg / ml. 2B 1 ml of 0.1 mol / L phosphate buffer 5.70 solution of pH 7, OiO, 5: rOfl 9 units / ml; peroxidase 1 unit act / ml; -AAA 0.050 mg / ml; phenol 0.65 mg / ml; LSA 10 mg / ml. 3B 1 ml of 0.1 mol / L phosphate buffer 6.60 solution of pH 7,, 5: YEAR 9 units / ml; peroxidase 1 unit / ml; -AAA 0.050 mg / ml; phenol 0.65 mg / ml; LSA 11 mg / ml.