CS270637B1 - Analytical means for the determination of diagnostically important components of biological fluids - Google Patents

Abstract

The solution relates to an analytical agent for determining diagnostically significant components of biological fluids based on enzyme reactions. The agent according to the solution contains as a stabilizing and/or separating agent propylene glycol ether of methylcellulose of general structure I, where R is H, CH^ or CH3CH(OH)CH2-radical, with methoxy groups being substituted preferably in 19 to 32% and hydroxypropoxy groups in 4 to 15%. Compared to the current state of the art, the agent according to the solution significantly increases the service life and quality parameters

Description

According to the invention, there is provided an agent for the solidification of important components of biological fluids based on enzymatic reactions. In contrast to the current known state of the art, this analytical composition contains a new stabilizing agent, which substantially increases the service life of the composition and its qualitative parameters.

In clinical-biochemical laboratory diagnostics, the vast majority of tests are currently based on enzyme methods. This is the case, for example, in the determination of glucose, uric acid, cholesterol or so-called triglycerides, where the relevant analytes are converted by means of specific enzymes - oxidoreductases (glucose oxidases, uricases, cholesterol oxidases or glycerophosphatoxidases) - into their oxidax products. The analytically very difficult determination of primary complex analytes is in this way converted into the determination of an incomparably simpler substance - hydrogen peroxide, for the determination of which its relatively strong oxidizing properties are used. The most commonly used procedure is the oxidation of a suitable leukobase redox indicator or chromogenic system based on oxidative coupling in the presence of a specific enzyme peroxidase to color oxidation products, the amount of which is equivalent to the amount of primary analyte. The special progressive field of diagnostics has recently been occupied mainly by dry phase diagnostic agents, such as powder mixtures, tablets, lyophilisates and especially reagent strips, in which all reagents needed for auxiliary reactions and final chromogen color reactions are contained in a suitable carrier in solid , resp. dry phase. With these systems, perfect separation of the individual incompatible components, stabilization of the labile components and protection against the sorption of atmospheric moisture is necessary, which reconstitutes the reaction system and starts undesired reactions, leading to the decomposition of the system. For the separation and stabilization of enzymes, predominantly hydrophilic polymeric substances of natural or synthetic origin are used, such as gelatin, agar, acacia, dextran, albumin, alginate, polyvinylpyrrolidone or polyvinyl alcohol, described for example in U.S. Pat. No. 3,814,668, U.S. Pat. No. 4,066,403 or methyl vinyl ether / maleic anhydride copolymer (U.S. Pat. No. 3,453,180). Protection of systems based on so-called dry chemistry from air humidity is achieved by thorough drying and hermetically sealed packaging, or. additionally provided with desiccants (silica gel, molecular sieves). Also described for reagent strips is a method of protecting the reaction system from the effects of atmospheric moisture, consisting of sealing the reagent paper between synthetic foils in such a way that the liquid to be examined can penetrate the reagent zone only through lateral exposed edges (DOS δ. 1 546 307) or through a fine a polymeric network of polyamide, polyester, polyethylene, polyvinyl chloride or polyacrylonitrile as described in U.S. Pat. No. 3,802,842. U.S. Pat. No. 4,046,513 discloses a diagnostic composition wherein the individual agents are applied (printed) in the form of separate, non-overlapping dots. The reagents mix with each other only after soaking the strip in the examined liquid. However, this method is technologically difficult and has not been applied in practice.

These methods of system protection have a number of disadvantages, such as uneven coloration of zones due to liquid diffusion, undesirable so-called edge effects, insufficient system stabilization, complex technology, etc. When using conventional protective colloids, eg gelatin, agar, it remains bound in the matrix residual moisture, which reduces the stability of the reaction system and also provides a suitable medium for the undesired growth of microorganisms. Somewhat more advantageous in this respect appears to be the use of synthetic hydrophilic polymers, e.g. polyvinylpyrrolidone or polyvinyl alcohol, which have stabilizing effects of enzymes and separate the individual components of the reaction system. However, said polymers also have a number of disadvantages, e.g. they contain residues of peroxide-based radical polymerization initiators (e.g. dibenzoyl peroxide, cumyl hydroperoxide), which have an adverse effect on the stability of the reaction system and also show a fairly high sorption of atmospheric moisture.

CS 270637 Bl

These shortcomings are eliminated by the present solution, the subject of which is an analytical agent based on enzymatic reactions. Its essence lies in the fact that it contains propylene glycol ether of methylcellulose of general structure I as a stabilizing and / or separating agent.

(I), wherein R is H-, CH 2 - or CH ₂ CH (OH) CH 2 - radical, the methoxy groups being preferably substituted in 19 to 32% and the hydroxypropoxy groups in 4 to 15%. This polymer is an excellent protective colloid and stabilizing agent with other favorable properties of the adhesive, filler and tabletting medium. It is highly resistant to bacterial contamination and inert to enzymes and commonly used reagents. It has excellent and fast solubility in water even at normal temperatures and in organic solvents and provides clear solutions. Moisture sorption depending on relative humidity is 3 to 4 times lower than, for example, the most commonly used polyvinylpyrrolidone (see Fig. 1)

relative humidity /%/

When applied to the reagent strips, the resulting color product is also affinity for the gel, increasing the brilliance, homogeneity and stability of the resulting color of the indicator zone, which is important both for the visual strips and especially for the strips evaluated reflectometrically. Thus, propylene glycol ether of methylcellulose appears to be a significant protective and stabilizing agent of reaction systems, especially on the progressive basis of so-called dry chemistry, the application of which eliminates the above-mentioned disadvantages of existing compositions, mainly on the principle of sensitive enzyme reactions. Means

CS 270637 B1 according to the invention contains, in addition to the chromogenic system, enzymes and the above-mentioned stabilizer, other substances known per se and commonly used for similar purposes, such as dampers, accelerators, surfactants and other auxiliaries.

Example 1

Reagent strips for the determination of glucose in urine

Impregnation solution:

Citrate buffer, pH = 6.2 100 ml Ethanol ' 60 ml Acetone 45 ml Methylcellulose propylene glycol ether (Klucel) 4.3 g Glucose oxidase 100 kU Peroxidase 15 kU Gelatin 0.2 g Tartrazine, 1% aqueous solution 6 ml 1- (4-sulfophenyl) -3-methyl-5-pyrazolone 2.5 5 4-aninophenazone 1.6 g Benzyldimethylhexadecylammonium chloride 0.8 g

Filter paper weighing 180 g / m is impregnated with the solution to saturation, dried with a stream of warm air and the reagent paper thus prepared is cut into 6x6 mm squares, which are glued to a plastic pad, in urine containing various concentrations of glucose. the strips provide a color gradation in the range of 0 to 170 mmol / l glucose after soaking. Unlike strips containing, for example, polyvinylpyrrolidone, the strips show about twice as high stability under the same storage conditions.

Example 2

Diagnostic tool for the determination of plasma glucose Reagent I: 4-aminoantipyrine 45 mmol Peroxidase 25 kU Glucose oxidase 300 kU Methyl cellulose propylene glycol ether (Pharmacoat 603) 3 G

The reagents are dissolved in 400 ml of distilled water and the resulting solution is lyophilized. The mixture is stable min. for 24 months

Reagent II: Citrate buffer (pH = 6) 0.2 mol

Primaquine diphosphate 0.01 mol

Sodium lauryl sulfate 0.7 mmol

Rain. water 300ml

Upon application, 1 part of reconstituted Reagent I is mixed with 100 parts of Reagent II, and this mixture of reagents is mixed with the sample in a ratio of 100: 1. The resulting color is directly proportional to the glucose concentration in the sample.

CS 270637 Bl

Example 3

Diagnostic strip for acid determination Impregnation solution I: Phosphate buffer (pH = 7) Uricase Methylhydroxypropylcellulose (Metolose) Peroxidase Methanol Distilled water Impregnation solution II: Methanol Dest. water 3-methyl-2-benzthiazolinone hydrazone hydrochloride 1- (4-sulfophenyl) -3-carboxy-5-pyrazolone benzyldimethyl-n-hexadexylammonium chloride

The indicator zones of the strip are prepared by successively applying and drying both impregnation solutions on filter paper weighing 120 g / m 2. Upon contact of the zone with a sample of biological fluid containing uric acid, a coloration occurs in proportion to the concentration of analyte in the sample.

urinary serum ml

AT

1.5 g

In ml ml ml ml

0.35 g

1.2 g

0.2 g

Example 4

Reagent strips for determination of blood glucose

Impregnation solution I:

3,5,5,5-tetramethylbenzidine dihydrochloride 12g

Sodium dioctylsulfosuccinate 1g

Methylcellulose propylene glycol ether (Klucel) 23g

Acetone 600ml

Ethanol 450ml

Distilled water 200ml

Impregnation solution II:

Citrate buffer (pH = 5.3) 300ml

Glucose oxidase 105kU

Peroxidase 200kU

Propylene glycol ether methylcellulose (Pharmacoat 606) 25g

Impregnation solution III.

Ethylcellulose 7, g

Toluene 200ml

CS 270637 B1 '5

Impregnation solutions I, II and III are successively applied to filter paper weighing 180 g / m 2, followed by drying. The reagent paper is then cut into 6 x 6 mm squares and glued to a plastic pad. The determination of glucose by means of these strips is carried out by applying a drop of blood to the indication zone of the strip, which is removed by rinsing with water after the incubation period. The resulting color of the strip is evaluated either semiquantitatively by comparison with a color calibration scale or reflectometrically. The strips, stored in well-sealed containers, are stable for at least three years. ·

Claims (1)

OBJECT OF THE INVENTION Analytical means for the determination of diagnostically important components of biological fluids which are converted into their oxidation products by means of specific enzymatic reactions, characterized in that it contains propylene glycol ether of methylcellulose of general structure I as a stabilizing and / or separating agent (i), wherein R is H, CH 2 or CH ₂ CH (OH) CH 2, a radical, the methoxy groups being preferably substituted in 19 to 32% and the hydroxypropoxy groups in 4 to 15%.