RU2268253C1 - Composition for storage of 3,3',5,5'-tetramethylbenzidine hydrochloride and urea peroxide aqueous solution - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention proposes the development of composition for storage of 3,3',5,5'-tetramethylbenzidine hydrochloride and urea peroxide solution that comprises the following components, g/l: 3,3',5,5'-tetramethylbenzidine hydrochloride, 0.1-0.4; urea peroxide, 0.1-1.7; hydroxypropyl-β-cyclodextrin, 2-5; polyvinyl alcohol, 1-4; procline 150, 0.05; 2-mercaptoethylamine hydrochloride, 0.007-0.03; glycerol, 62-500 in citrate buffer medium with pH 3.77 that can comprise orcein monohydrate additionally in the concentration 0.004-0.02 g/l. Technical result involves the development of such substrate solutions representing the main component of diagnostic immunoenzyme sets when substrate as components of sets are monocomponent and stable that allows avoiding additional stage in carrying out analysis, namely, mixing two components of 3,3',5,5'-tetramethylbenzidine hydrochloride and urea peroxide before onset of assay of enzymatic activity, and to use the same set for prolonged time. Invention can be used for simultaneous storage of 3,3',5,5'-tetramethylbenzidine hydrochloride and urea peroxide aqueous solution used as a substrate mixture ready for using, for example, in immunoenzyme analysis.

EFFECT: valuable biochemical and analytical properties of composition.

2 cl, 2 tbl

Description

The invention relates to the field of biochemistry and is used for simultaneous storage of an aqueous solution of 3.3 ', 5.5'-tetramethylbenzidine hydrochloride with urea peroxide, used as a substrate mixture, ready for use, for example, in an enzyme-linked immunosorbent assay (ELISA).

For the analysis, commercial test systems are usually used, including a complete set of reagents, sufficient to simultaneously conduct several dozen analyzes. The main components of the kits are polystyrene plates with immobilized antibodies or antigens, conjugates of antibodies or antigen with an enzyme, standards for the analyte, components of buffer solutions, enzyme label substrates. Most commercial kits use peroxidase as the enzyme label. During the reaction catalyzed by peroxidase, 3.3 ', 5,5'-tetramethylbenzidine is oxidized to a product with a blue color, which turns yellow when the enzymatic reaction is stopped by the addition of a stop solution (0.2 M sulfuric acid solution), with absorption maximum at a wavelength of 450 nm.

In standard reagent kits, the substrate solution is generally supplied in a stable two-component form, i.e. in one bottle a solution of 3.3 ', 5.5'-tetramethylbenzidine in an organic solvent and separately in another bottle a buffer solution of hydrogen peroxide. However, after the preparation of the substrate mixture by mixing the two reagents, the solution quickly loses activity, while the blue quickly. All this necessitates its rapid use. In practice, however, often the need arises to pose only a small number of analyzes.

Substrate solutions are the main components of diagnostic enzyme immunoassays. It is preferable when the substrates included in the kits are one-component and stable, i.e. are ready to use. This allows you to avoid performing an additional step during the analysis - mixing two components - TMB and urea peroxide before starting the measurement of enzymatic activity and use the same set for a long time.

3.3 ', 5.5'-Tetramethylbenzidine is a non-polar organic water insoluble substance. Various organic solvents are known for dissolving 3,3 ', 5,5'-tetramethylbenzidine molecules, such as N-methylpyrrolidone (NMP) [1], dimethyl sulfoxide (DMSO) [2], dimethylformamide (DMF) [3, 4, 6] , various alcohols with a molecular weight of from 40 to 100, i.e. ethanol [6], n-propanol, isopropanol, methoxypropanol [6], n-butanol, tert-butanol, as well as acetonitriles and ketones [5].

For environmental safety, toxic and volatile solvents are replaced with harmless components, which contributed to the dissolution of the hydrophobic molecule 3,3 ', 5,5'-tetramethylbenzidine in an aqueous solution. As such solvents, various surface-active substances (surfactants) are used, the molecules of which consist of two parts - hydrophilic and hydrophobic. The hydrophobic parts of surfactant molecules interact with a hydrophobic molecule, for example, 3,3 ', 5,5'-tetramethylbenzidine, surrounding it with a dense layer. As a result, the outer surface of such a coil (micelles) is formed by the hydrophilic ends of the surfactant molecule, which has an affinity for water. Thus, surfactant, without changing the structure of 3,3 ', 5,5'-tetramethylbenzidine, allows you to transfer it to water, the resulting system is called dispersion. Over time, coagulation begins in the dispersion system (gradual coalescence of hydrophobic particles), leading to the destruction of the lyophilic colloidal system. For stability of the thermodynamic dispersion per unit of hydrophobic substance, a large number of suitable different surfactants were selected in earlier studies.

Various surfactants are known, for example, water-soluble polymers - polyethylene glycol, polyethylene oxide, polyvinylpyrrolidone [7, 11], acrylamide polymers, polymers of acrylic and methacrylic acids, [8], polyvinyl alcohol (PVA) [10, 12], also cyclic oligosaccharides, for example β β-cyclodextrins [2, 9]. The cyclodextrin molecule consists of several glucoside rings joined into one large ring. Cyclodextrins easily interact with organic molecules to form inclusion complexes - complexes in which an organic hydrophobic molecule is located inside the cyclodextrin molecule. Such complexes are hydrophilic due to the external hydrophilic sphere of the cyclodextrin molecule.

All these surfactants allow you to create a stable dispersion system TMB and maintain its stability for a long time, which makes it possible to use this composition as a finished commercial product. The single component of the substrate avoids the stage of mixing the two components of the substrate before analysis, which reduces the analysis time. The absence of toxic and volatile organic solvents and the non-carcinogenicity of 3,3 ', 5,5'-tetramethylbenzidine creates safety for health and the environment when working with this substrate. High stability and single component substrate ensures its readiness for use as a commercial reagent. The readiness of the reagents for use is especially convenient for diagnostic laboratories performing a small number of analyzes daily, and allows significant savings of all reagents used in the diagnostic kit.

A composition for storing an aqueous solution of 3.3 ', 5.5'-tetramethylbenzidine with hydrogen peroxide, ready for use, in which hydroxypropyl-β-cyclodextrin (GPβC) (0.004-0.01 g / l) and a lyophilic additive - 1,2,6-trihydroxyhexane (0,042-0,6 g / l) [13].

The objective of the invention is to provide a composition for storing an aqueous solution of 3.3 ', 5.5'-tetramethylbenzidine hydrochloride with urea peroxide (PM), which consists in the selection of two types of components: surfactants to create a stable dispersion and antioxidants to prevent oxidative processes in aqueous a solution of TMB and urea peroxide.

This problem is solved by the proposed composition. Its main components include polyvinyl alcohol, hydroxypropyl-β-cyclodextrin, glycerin (GL), orcinum monohydrate (MO), 2-mercaptoethylamine hydrochloride (2-MEA) in 0.1 M citrate buffer pH 3.8.

The hydrophilic inclusion complexes of the 3.3 ', 5,5'-tetramethylbenzidine molecule with the hydroxypropyl-β-cyclodextrin molecule are distributed into a gel network formed from the polyvinyl alcohol polymer chains and create a lyophilic colloidal system.

Glycerin is introduced into the system as a lyophilic additive, which allows to increase the stability of the dispersion, preventing its delamination.

2-mercaptoethylamine hydrochloride and orcine monohydrate were used as antioxidants that react with free radicals and prevent the oxidation of 3,3 ', 5,5'-tetramethylbenzidine molecules.

To prevent the negative influence of the microflora of the medium on the stability of the drug, proclin 150 (PR150) is added to the substrate mixture as an antibacterial agent. The content of proclin 150 in the mixture does not exceed 0.05 g / l, since a larger amount of it has an inhibitory effect on peroxidase.

Using the proposed composition allows you to store the substrate solution for determining peroxidase based on 3.3 ', 5,5'-tetramethylbenzidine hydrochloride with urea peroxide, in a ready-to-use form.

The invention is illustrated by example.

Example

Substrate solutions are prepared by the sequential addition of polyvinyl alcohol, hydroxypropyl-β-cyclodextrin, glycerol, proclin 150, 2-mercaptoethylamine hydrochloride, orcine monohydrate, 3.3 ', 5.5'-tetramethylbenzidine hydrochloride, urea peroxide in an aqueous solution of nitrate buffer pH 3 77 with different ratios of concentrations (table 1, 2). The activity of the TMB solution is determined by the enzymatic activity of the conjugate of antispecies antibodies with horseradish root peroxidase (AT-Px) (the concentration of the conjugate 1 × 10 ^-8 M is determined by its enzymatic activity), and is measured photometrically as follows. In a well of a 96-well polystyrene tablet sensitized with rabbit antibodies, 100 μl of conjugate solution was added, incubated for 1 hour at T = 37 ° C and then washed with phosphate buffer pH 7.4. Then, 100 μl of a sample of the substrate solution is added and incubated for 15 minutes. In the reaction catalyzed by peroxidase, the oxidation of 3,3 ', 5,5'-tetramethylbenzidine dihydrochloride occurs in the product having a blue color. The enzymatic reaction is stopped by the addition of 0.2 M sulfuric acid solution and the absorbance of the solution in the well is measured at λ = 450 nm.

The initial activity of the solutions of TMB substrates is presented in table 1.

The selected samples are thermostated at T = 37 ° C for 30 days and the activity of the TMB substrate is measured in them as they are inactivated by the method described above.

The ratio of TMB activity at a given time of inactivation to its initial activity characterizes the relative activity of a TMB solution (table 2).

Bibliography

1. US patent No. 4596770, 1986.

2. US Patent No. 5792618, 1998.

3. US patent No. 5006461, 1991.

4. US patent No. 5013646, 1991.

5. U.S. Patent No. 5,176,999, 1993.

6. Patent Canada No. 1290661, 1991.

7. US Patent No. 4615972, 1986.

8. US Patent No. 5047318, 1991.

9. Patent Canada No. 1277895, 1990.

10. Patent Canada No. 1281644, 1991.

11. US patent No. 4615972, 1986.

12. US Patent No. 6221624, 2001.

13. US patent No. 5792618, 1998.

Table 1. The content of substances in the samples, g / l The initial activity of the substrate TMB, D ₄₅₀ TMB PM PR150 PVA GPβC GL 2-MEA MO 0.12 0.157 0.05 1.28 2,56 62.5 0.0073 0.007 0.828 0.16 0.157 0.05 1.28 2,56 62.5 0.0073 0.007 1.12 0.18 0.157 0.05 1.28 2,56 62.5 0.0073 0.007 1,413 0.2 0.157 0.05 1.28 2,56 62.5 0.0073 0.007 1,685 0.26 0.157 0.05 1.28 2,56 62.5 0.0073 0.007 1.9 0.32 0.157 0.05 1.28 2,56 62.5 0.0073 0.007 2,265 0.38 0.157 0.05 1.28 2,56 62.5 0.0073 0.007 2,413 0.38 0.4 0.05 1.28 2,56 62.5 0.0073 0.007 2,642 0.38 0.645 0.05 1.28 2,56 62.5 0.0073 0.007 2,587 0.38 0.89 0.05 1.28 2,56 62.5 0.0073 0.007 2,524 0.38 1,137 0.05 1.28 2,56 62.5 0.0073 0.007 2,264 0.38 1,382 0.05 1.28 2,56 62.5 0.0073 0.007 2,031 0.38 1,626 0.05 1.28 2,56 62.5 0.0073 0.007 1,791 0.38 0.157 0.05 1.28 2,56 125 0.0073 0.007 1,856 0.38 0.157 0.05 1.28 2,56 250 0.0073 0.007 0.785 0.38 0.157 0.05 1.28 2,56 500 0.0073 0.007 0.407 0.38 0.157 0.05 1.28 2,56 62.5 - 0.007 2.98 0.38 0.157 0.05 1.28 2,56 62.5 0.0146 0.007 1,742 0.38 0.157 0.05 1.28 2,56 62.5 0,0219 0.007 1,145 0.38 0.157 0.05 1.28 2,56 62.5 0,0292 0.007 0.736 0.38 0.157 0.05 1.28 2,56 62.5 0.0073 - 2,782 0.38 0.157 0.05 1.28 2,56 62.5 0.0073 0.0045 2,629 0.38 0.157 0.05 1.28 2,56 62.5 0.0073 0.01 2,299 0.38 0.157 0.05 1.28 2,56 62.5 0.0073 0.015 2,052 0.38 0.157 0.05 1.28 2,56 62.5 0.0073 0.0045 2,629 0.38 0.157 0.05 1.28 2,56 62.5 0.0073 0.01 2,299 0.38 0.157 0.05 1.28 2,56 62.5 0.0073 0.015 2,052

Table 2. The content of substances in the samples, g / l The relative activity of the substrate TMB,% Time days TMB PM PR150 PVA GPβC GL 2-MEA MO 0 7 fourteen 21 31 0.38 0.157 - 1.28 2,56 - - - one hundred twenty 7 3 one 0.38 0.157 0.05 1.28 2,56 - - - one hundred twenty 17 13 10 0.38 0.157 0.05 1.28 2,56 62.5 - - one hundred 45 36 twenty fifteen 0.38 0.4 0.05 1.28 2,56 62.5 0.0073 0.007 one hundred 90 87 83 78 0.38 0.157 0.05 1.28 2,56 62.5 0.0073 - one hundred 90 88 85 75 0.38 0.157 0.05 1.28 2,56 62.5 0.0073 0.0045 one hundred 92 89 87 80 0.38 0.157 0.05 1.28 2,56 62.5 0.0073 0.007 one hundred 96 95 90 87 0.38 0.157 0.05 1.28 2,56 62.5 - 0.007 one hundred 60 54 49 40

Claims (2)

1. Composition for storing an aqueous solution of 3.3 ', 5.5'-tetramethylbenzidine hydrochloride and urea peroxide, containing hydroxypropyl-β-cyclodextrin and a lyophilic additive, characterized in that the composition includes polyvinyl alcohol, proclin 150, hydrochloride 2- mercaptoethiamine, and glycerol is used as a lyophilic additive with the following components: 3.3 ', 5.5'-tetramethylbenzidine hydrochloride 0.1-0.4 g / l; urea peroxide 0.1-1.7 g / l; hydroxypropyl-β-cyclodextrin 2-5 g / l; polyvinyl alcohol 1-4 g / l; proklin 150 0,05 g / l; 2-mercaptoethylamine hydrochloride 0.007-0.03 g / l; glycerol 62-500 g / l; in a medium of citrate buffer with a pH of 3.77. 2. The composition according to claim 1, characterized in that it additionally contains 0.004-0.02 g / l of Orcine monohydrate.