LT4121B - Kit for the cholesterol vizualization on the skin and process for preparing conjugate for use in cholesterol determination - Google Patents

Abstract

The offered kit for visual detection of cholesterol on skin surface consists of a conjugate related to epidermis cholesterol, and a developer solution, which contains of the following ingredients (mg/ml):citric acid: 1,000 to 2,000;3.3',5.5'- tetra-methyl-4.4'-diamynodiphenile: 70 to 83;hydrogen peroxide: 250 to 300;disodium ethylene- diamine tetraacetate: 300 to 400;disodium dihydrogen pyrophosphate: 15 to 30;acetanilide: 5 to 30;water remaining part up to 1 litre.It is recommended to produce the conjugate, which is related to epidermis cholesterol, having initially treated N- vinylpyrrolidone and maleine acid anhydride copolymer with digitonine, and then having combined the ferment peroxidase modified by treating it with glutaraldehyde and 1.6 hexamethylene diamino.

Description

BACKGROUND OF THE INVENTION The present invention relates to the field of biological and bioorganic chemistry, more particularly to diagnostic tools for the detection of disorders characterized by elevated cholesterol levels in the skin, such as atherosclerosis.

Kits for visual determination of cholesterol on the surface of the skin consist of 2 main components: a conjugate having affinity for epidermal cholesterol and a developing solution. The kit also contains auxiliary components: conjugate buffer, other excipients, pipettes and the like.

Kits for visual setting have become very popular in recent years. They have the advantage of being quick, easy and obvious to identify a particular material.

Typically, for visual determination of cholesterol, the conjugate is dissolved in buffer and a drop of the resulting solution is applied to the skin of the palm of the hand. After a fixed period of time (e.g., 1 min), the excess conjugate is washed with water and the amount of conjugate bound to palm cholesterol is evaluated by adding a drop of developer solution according to the color spot formation and its intensity.

The main component of the set. - The closest method for preparing a conjugate having epidermal cholesterol affinity is described in USSR SU 1675769. The present invention describes a substantially similar method for preparing a conjugate having epidermal cholesterol affinity by attaching to the N-vinylpyrrolidone and maleic anhydride copolymer at the beginning of the enzyme digonoxide. Unfortunately, the conjugate thus obtained is not very stable over long periods of time.

The composition of the other essential component of the kit, the developer solution, is known, for example, from the review [Yu. M. Lopukhin, The Skin and Atherosclerosis,

Physicochemical Aspects of Medicine, 1992, vol. 3, part 4, p. 46-501]. It is an aqueous solution containing N, N-diethyl-p-phenylenediamine sulfate, hydrogen peroxide, sodium thiosulfate, stabilizing solution and potassium bromide.

This developer solution is unstable and can only be used (remains colorless) for 6-8 hours.

These shortcomings significantly limit the clinical use of the entire kit.

For industrial kits, it is essential that the kits retain their functional properties for a sufficient period of time (eg 6 months) to transport, store, and use the kit. The present invention solves this problem by using the formulation of the present invention.

In order to improve the stability of the components and of the kit as a whole, it is proposed that a solution of the following components be prepared in water in the following amounts (mg / l): citric acid

3,3 ', 5,5'-tetramethyl-4,4'-diaminodiphenyl hydrogen peroxide disodium ethylenediaminetetraacetate disodium dihydrogen pyrophosphate acetanilide

1000 - 2000 70 - 83

250-300

300-400

- 30

- 30

It is proposed to produce a conjugate having affinity for epidermal cholesterol by reacting a copolymer of N-vinylpyrrolidone and maleic anhydride initially with digitonin followed by coupling with the enzyme peroxidase modified by glutaraldehyde and 1,6-hexamethylenediamine.

Example 1.

The kit consists of the following components:

- Conjugate i.e. A copolymer of N-vinylpyrrolidine and maleic anhydride with digitonin and peroxidase attached or modified by the action of glutaraldehyde and 1,6-hexamethylenediamine on peroxidase;

- developer solution;

- buffer solution for conjugate solution.

Prepare the solution as follows:

Citric acid is dissolved in distilled water, concentrated hydrochloric acid is added to pH 2.5, dissolved in 3,3 ', 5,5'-tetramethyl-4,4'-diaminodiphenyl, 10% sodium hydroxide solution is added to pH 3.5 and the 90% of the total disodium ethylenediaminetetraacetate is dissolved in the mixture. Separately prepare a solution containing hydrogen peroxide, 10% total disodium ethylenediaminetetraacetate, disodium dihydrogen pyrophosphate and acetanilide. Mix both solutions well. The influence of the composition of the solution on its stability is illustrated in the following table.

The peroxidase activity of the conjugate with modified peroxidase stabilizes the peroxidase activity. This is very important for the lifetime of the kit since the stability term for both components is virtually equal.

Table

Component concentrations, mg / 1 Stability * solution_month.

No.

A B C D E F

1 1600 83 283 344 22nd 13th > 6th 2 2000 83 283 344 22nd 13th 6th 3 1000 83 283 344 22nd 13th 6th 4 1600 70 283 344 22nd 13th > 6th 5 1600 83 300 344 22nd 13th 6th 6th 1600 83 250 344 22nd 13th > 6th 7th 1600 83 283 400 22nd 13th > 6th 8th 1600 83 283 300 22nd 13th 6th 9th 1600 83 283 344 30th 13th > 6th 10th 1600 83 283 344 15th 13th 6th 11th 1600 83 283 344 22nd 30th > 6th 12th 1600 83 283 344 22nd 5 6th

where A is citric acid

B - 3,3 ', 5,5'-tetramethyl-4,4'-diaminodiphenyl C - hydrogen peroxide

D - Disodium ethylenediaminetetraacetate E - Disodium dihydrogen pyrophosphate F - Acetanilide * The time during which the color intensity of the solution, measured with a spectrophotometer at 652 nm in the control sample, does not exceed 0.05.

Example 2: Method for Preparation of Conjugate

Dissolve 200 mg of peroxidase in 15 ml of 0.2 M phosphate buffer, pH 7.0. To the solution are added dropwise 9.0 ml of 25% glutaraldehyde solution. The mixture is stirred for 24 hours and then washed 6 times with 0.2 M phosphate buffer pH 7.0 in an ultrafiltration apparatus. Then 1 ml of a 30% aqueous solution of 1,6-hexamethylenediamine is added to the solution and the mixture is stirred for 24 hours. The reaction mixture is then washed 8 times with 0.2 M phosphate buffer in an ultrafiltration apparatus. The resulting solution of modified peroxidase is incubated with N-vinylpyrrolidone and maleic anhydride copolymer to which digitonin is attached. The resulting conjugate retains more 80% of the peroxidase activity for 6 months.

The use of the present invention provides the following advantages:

1. The stability of the proposed developer (the time during which the solution remains colorless) increases from 6-8 hours to 6 months, i.e. 540-720 times, and the stability of the conjugate (the time to maintain at least 80% of the peroxidase activity) increases from 2-3 months. up to 6 months, i.e. 2-3 times.

2. Operational properties of conjugate peroxidase activity and stabilizer solution stabilization.

improves the set

Claims (2)

DEFINITION OF INVENTION 1. Kit for visual determination of cholesterol on the surface of the skin, consisting of a conjugate having affinity for epidermal cholesterol and a developer solution, characterized in that the developer consists of (mg / l): citric acid 1000-2000 3,3 ', 5,5'-tetramethyl-4,4'-diaminodiphenyl 70-83 hydrogen peroxide 250-300 disodium ethylenediaminetetraacetate 300-400 disodium dihydrogen pyrophosphate 15-30 acetanilide 5-30 water up to 1 1 2. A process for the preparation of a conjugate having an affinity for epidermal cholesterol by attaching digitonin to a copolymer of N-vinylpyrrolidone and maleic anhydride followed by peroxidase modification by the action of glutaraldehyde and 1,6-hexamethylenediamine.