DE2415257C2 - Diagnostic agent for the detection of glucose in urine - Google Patents

Description

The invention relates to a diagnostic agent for the detection of glucose in urine, consisting of an absorbent carrier containing glucose oxidase, peroxidase, an indicator and a buffer is impregnated.

The detection of glucose in the urine is of great importance for diagnosis and monitoring of the disease Diabetes. Because of this fact, there are a large number of so-called rapid tests in the patent literature some of which are on sale. They are based almost entirely on the following Principle: Glucose is converted into gluconic acid and hydrogen peroxide by means of glucose oxidase and oxygen oxidized, this oxidizes by means of peroxidase an indicator to a f-like compound. Although a A number of such indicators is described, so far mainly o-To) idin has been used.

In general, a pH range of 4 to 7 is described for such a glucose test, starting with the usual buffers such as phosphate, citrate and similar buffers. However, it is preferred almost exclusively pH 5, as sensitivity at this pH value and gradation are obviously optimal (see e.g. U.S. Patent Nos. 3,050,373 and 28 48 308 and DT-PS 15 98 809). However, test papers with this pH have the serious one Disadvantage that the reaction color is weakened in so-called "diabetic ketoacidosis" is ia can even be completely suppressed. In the Ketoacidosis, larger amounts of acetoacetic acid and hydroxybutyric acid occur in the urine, which causes the Reaction of the test papers with a buffer of pH 5 interferes. Ketoacidosis occurs mainly in diabetes

ίο on "but also with other diseases and with Hunger states. "Diabetic ketoacidosis" only differs from the others in that simultaneous occurrence of glucose in the urine. A quick test for glucose, which interferes with ketoacidosis

is is therefore neither for differential diagnosis of Keto'acidose is still ideally suited for the diagnosis and monitoring of the course of diabetes.

Our »experiments have shown that by increasing the pH of the glucose test

papers to 5.8 to 6.2, preferably 6.0 the disorder can reduce ketoacidosis. When using conventional buffers, however, show that the gradation of the color reaction deteriorates greatly without the disturbance being avoided in all cases. In addition, the durability of the test papers in many cases much to be desired.

It has now surprisingly been found that glucose test papers are obtained which are sensitive, react quickly and with good gradation and which are not disturbed in ketoacidosis if you are in a diagnostic agent of the type mentioned above as a buffer 2- (N-morpholino) ethanesulfonic acid (MES) used. These papers are also completely stable at long-term thermal loads, what

is of particular importance when used in the tropics.

2- (N-Morpholino) -ethanesulfonic acid is indeed a known buffer; it has been reviewed by Good, Biochemistry 5 (1966), p. 467, for work with biological

Systems introduced, but only in liquid form Phase and not in solid reagent preparations such as test papers. For those skilled in the art, these were surprising Effects of this special buffer when used in glucose test papers are not without further predictable, especially since other buffers used in biological systems, such as e.g. B. the so-called Tris, did not show any effects corresponding to the MES (see the following table).

Buffer (pH 6.0) Interference caused by 250 mg% decrease in reaction Reaction color with 100 to 1000 mg% acetoacetic acid and after 30 days at 6O ⁰ C Glucose in urine 2000 mg% ^ -hydroxy

butyric acid in the urine

MES, 0.5 m
(see example 1)

Phosphate, 0.25 m
Citrate, 0.25 m
Citrate, 0.5 m
Tris citrate, 0.5 m
Imidazo !, 0.25 m
3-aminopyridine, 0.5 m
Malonate, 0.5 m
Maleate, 0.125 m
Pyrophosphate, 0.25 m

Comparison with pH 5:
Citrate, 0.25 molar

light green to blue black

light green to gray green
light green to gray green light green to dark green light green to green
light green to gray green
green-yellow to light green
gray-green to dark green light green to gray-green
light green to dark green

dark green to blue black no disturbance

moderate disorder
moderate disorder
moderate disorder
minor disturbance
strong disorder
minor disturbance
moderate disorder
moderate disorder
moderate disorder

strong disorder

practically no waste

very heavy drop
heavy drop
heavy drop
heavy drop
very heavy drop
no more reaction
heavy drop
moderate drop
very heavy drop

little waste

3 4

As can be seen from the table, only MES glucose oxidase provides 222 mg

as a buffer substance optimal test peroxidase in every respect 28 mg

papers. With the rest of the common and also MES buffers

Unusual buffer substances give test (pH 6.0; 1.0 M aqueous solution) ... 50 ml

papers showing a sometimes considerable disruption from 5 tartrazine 80 mg

Acetoacetic acid and hydroxybutyric acid (i.e. for o-tolidine 420 mg

Ketoacidosis) and also less recommended Ethanol 33 ml

fragile and / or less durable. Water, distilled to 100 ml

The MES buffer used in the present invention is

in concentrations of 25 to 75 mmol, preferred io The test paper reacts with urines from 50 to

wise 5OmMoI, contain 1000 mg% glucose per 100 ml impregnation solution, with light green to

set. The desired pH range of bluish-black shades of good color gradation is obtained.

5.8 to 6.2, preferably 6.0, if you have the aqueous urine, the additional 25ümg% acetoacetate and about

Slurry of 2- (N-Morpholino) -ethanesulfone- containing 2000 mg% /? - Hydroxxybutyric acid, show

acid with the necessary amount of concentrated sodium »5 practically the same color reactions. No change

lye added. In addition, results in the desired pH range are observed after the test

a homogeneous solution. In addition to glucose oxidase, paper 15 days at 6O ⁰ C or 4 months at 40 ⁰ C

Peroxidase and an indicator (preferably o-Toli- has been stored, din), which are used in the usual concentrations

ao B e i s ρ i e 1 2 Intermediary (e.g. polyvinylpyrrolidone or polyethylene

glycol) and wetting agent (e.g. sodium lauroyl sarcosinate filter paper (Whatman No. 6) is treated with a

or sodium lauryl sulfate) soaked in the preparation of the solution of the following composition and

Find test papers use. Likewise, a dried at 5O ⁰ C: z of yellow dyes (additional example tartrazine or 35th

Naphtholgelb S) appropriate because it contains the colorant glucose oxidase 222 mg

make the stroke more clearly visible. Peroxidase 28 mg

Filter MES buffers are primarily used as absorbent carriers

papers in question, but also fleeces and felts (pH 5.8; 1.0 m aqueous solution) ... 65 ml

Cellulose or plastics can be used. 30 tartrazine 80 mg

For the production of the new diagnostic agent polyvinylpyrrolidone 300 mg

becomes in a known manner an absorbent carrier, pre-o-tolidine 420 mg

preferably filter paper, soaked with a solution, ethanol 30 ml

which contains the above ingredients, water, distilled to 100 ml

and then dried. 35

As a solvent for the impregnation solution, the test paper has practically the same properties

especially mixtures of water and volatile shafts like the test paper according to example 1. organic solvents, e.g. B. lower alcohols,

in question. Example 3

The test strips obtained in this way can 40

to be used as such, after being put into strips of filter paper (Schleicher and Schüll 595) using

are cut. If desired, a solution of the following composition can be soaked

Paper strips but also between plastic films and dried at 50 ° C:

seal in (cf. DT-PS 15 46 307) or on such glucose oxidase 222 mg

Seal or stick on foils; preferably 45 peroxidase is 28 mg

between a plastic film and a j ^ gg buffer

fine-meshed network according to DT-PS 2118455 (pH 6.2; 1.0 m aqueous solution) ... 25 ml

sealed. Sodium Lauroyl Sarcosinate 100 mg

In the following examples the invention becomes Naphthol Yellow S 100 mg

explained in more detail. 50 o-tolidine 420 mg

Example 1 Ethanol ■ · · 33 ml

". _u . , "..... _o . ...., "" _XTr , _x . . Water, distilled to 100 ml

Filter paper (Schleicher and Schüll 597 NF)

with a solution of the following composition, this test paper also has practically the same properties

soaked and ^{dried at 50 0} C: 55 properties like the test paper according to the example

Claims (3)

Patent claims: 1. Diagnostic means for the detection of glucose in urine, consisting of an absorbent Carrier that impregnates with glucose oxidase, peroxidase, an indicator and a buffer is, characterized in that the buffer used is 2- (N-morpholino) ethanesulfonic acid used. 2. Diagnostic agent according to claim 1, characterized in that the pH of the Buffer is between 5.8 and 6.2. 3. Diagnostic agent according to claim 2, characterized in that the pH of the Buffer is 6.0.