FI57025B - TESTING FOR THE PURPOSE OF A BLOOD AND A PERCENTAGE COMPARTMENT SUBSTANCER FOR CROPPING - Google Patents

Description

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(22) Hakamltptivi - AntBknlngtdag 13.12.7k

(23) AlkupUvi — Glltifhattdag 13.12.7U

(41) Became Public - Blivlt offwttlig 21.06.75

Patent and Registration Office .... ..... ,. . ,. ,,,, ..

• (44) Date of issue of the letter. -

Patent · och registerstyrelsen An »ek» n uttajd och utl.ikrift * n publkcnd 31 · 01.80 (32) (33) (31) «Watty atuoikaus-Begird priority 20.12.73

Federal Republic of Germany Förbundsrepubliken Tyskland (DE) P 23633 ^ .5 (71) Boehringer Mannheim GmbH., Mannheim-Waldhof, Federal Republic of Germany Förbundsrepubliken Tyskland (DE) (72) Walter Rittersdorf, Mannheim-Waldhof, Werner Giithlein, Mernheim Giithlein Hans-Georg Rey, Mannheim-Waldhof, Peter Rieckmann, Mannheim-Waldhof, Federal Republic of Germany-Förbundsrepubliken Tyskland (EE) (7 * 0 Berggren Oy Ab (5¼) Test strip for the detection of blood and other peroxidatively active substances in body fluids - Testremsa för pävisning av blod och andra peroxi-datiskt verksamma substanser i kroppsvätskor

The present invention relates to sensitive test strips for detecting very small amounts of blood and other peroxidatively active substances in body fluids.

Demonstration of small, invisibly visible amounts of blood in urine, feces, or vomiting is very important to detect bleeding in the stomach, intestines, and urinary tract. Such bleeding is caused, for example, by tumors, ulcers and inflammation in similar organs. In addition, certain hemolytic toxins may also cause free hemoglobin in urine and plasma. Blood and hemoglobin are peroxidatively active, i. release oxygen from the hydroperoxides and transfer it to specific acceptors. Similarly, peroxidatively active myoglobin is present in the urine, for example, after a heart attack. Blood in the urine is also very common due to bladder stones and kidney stones.

Their peroxidative effect is excellent for the sensitive detection of all these substances. The oxygen 2 5 / O 2 £ released from the hydrogen peroxide is then transferred to the chromogen, which is oxidized to a dye and thus indicates the presence of a peroxidatively effective compound. In particular, to demonstrate blood, this reaction has long been used in medical and forensic analytics. It is usually carried out by means of a reagent glass or drop reaction, in which case hydrogen peroxide is usually used as the hydroperoxide. Suitable chromogenic agents are, in particular, benzidine, o-tolidine or leukomalacite green.

The fastest are absorbent carriers, such as papers, which are impregnated with all the reagents necessary for the detection reaction and which, after simple immersion in body fluid, cause a color reaction. In keeping with the great importance that rapid experiments have recently achieved, a wide variety of such experiments have been developed to detect blood in body fluids.

Since the sensitivity of the rapid test is crucial for the detection of blood, attempts have already been made to improve the sensitivity of the detection reactions known per se by means of various additives. Thus, for example, German Offenlegungsschrift No. 1,242,905 describes test papers which contain certain quinoline derivatives, preferably quinine, as activating additives. Although it has been argued that sensitivity can be significantly increased by the additives shown, our own experiments have shown that it is practically impossible to detect thus improved test strips, e.g., blood at 5 Ery / mm ^, between.

It has now been found that test strips with hitherto unattainable sensitivity are obtained for the detection of peroxidatively active substances when a vinylpyridine of the general formula I is used as activator.

wherein the formula is a pyridyl group or a phenyl group optionally substituted by lower alkyl or alkoxy groups, and R 2 is hydrogen or a lower alkyl group.

3 b 7 O 2 6

Most preferred are compounds wherein = hydrogen. Lower alkyl and alkoxy groups have 1 to 4 carbon atoms and are preferably methyl or methoxy groups.

The present invention thus relates to test strips for detecting peroxidatively active substances in body fluids formed by absorbent carriers impregnated with hydroperoxide and at least one chromogen, characterized in that the carrier additionally contains a compound of the general formula I as activator.

Surprisingly, the compounds I do not increase the sensitivity of peroxidases of plant origin, such as, for example, horseradish peroxidase, but act specifically on the peroxidatively active compounds of human or animal organisms. Thus, it is possible to selectively detect blood or blood particles in the faeces or vomiting in addition to vegetable peroxidases. Thus, myoglobin can be detected with approximately the same sensitivity as hemoglobin.

The sensitivity of the detection reaction is increased by the various compounds of the general formula I, so that it is even possible in the urine to detect individual erythrocytes which appear on the test paper as colored dots. Thus, for example, ac-styrylpyridine can be used to prepare a test paper which can clearly detect a further 5 erythrocytes / mm of urine. This corresponds to a blood dilution of 1: 1,000,000.

It is clear that not all compounds of general formula I have the same activating properties. Thus, it is possible to adjust, for example, the sensitivity of the blood test in a completely practical manner.

Thus, for example, when test strips with increasing activity are added as activators, the compounds listed below are listed in the order in which they are listed: 4,57025 2- (phenylvinyl) -4-methylpyridine pyridyl-2-pyridyl-4-ethylene 2- (4-methylphenylvinyl) pyridine 2- (4-methoxyphenyl) ) pyridine di (pyridyl-2) ethylene pyridyl-2-pyridyl-3-ethylene α-styryl-pyridine

About 0.05-1.0 g, preferably 0.2-0.5 g, of the activating agent according to the invention are added per 100 ml of impregnation solution.

Other components of the rapid blood test include an organic hydroperoxide, an oxidation indicator, a buffer and a surfactant, and, if desired, a phosphoramide such as phosphoric acid trimorpholide for stabilization and other conventional excipients.

Suitable hydroperoxides are, for example, diisopropylbenzene hydroperoxide or 2,5-dimethylhexane-2,5-dihydroperoxide, indicators are those of the benzidine series, such as o-tolidine, or those of the heterocyclic azines, such as bis- (N-ethylquinolone-2) -azine or (N-methylbenzothiazolon-2 -) - (1-ethyl-3-phenyl-5-methyl-triazolon-2) -azine, as disclosed in German Patent No. 1,648,840.

The indicators are added in amounts of 0.05 to 5 g, preferably 0.2 to 1.0 g, per 100 ml of impregnation solution.

Suitable buffers are, for example, citrate, phosphate, phthalate or succinate buffers, in which case the pH and capacity must be chosen so that after immersion of the test strip in body fluid, this pH is 4-7, preferably 5-6.

It is also preferred to add small amounts (about 0.05-0.5 g per 100 ml) of a complexing agent such as sodium metaphosphate or ethylenediaminetetraacetic acid to the composition to avoid false positive reactions that may be caused by negligible amounts of metal.

Because of the relatively large amounts of water-soluble substances, the test papers tend to "break through", it is practical to add a thickener such as methylcellulose, and especially polyvinylpyrrolidone, to the composition in amounts of about 0.5-5 g per 100 ml.

Suitable wetting agents are suitably long-chain organic sulphates or sulphonates, such as, for example, sodium dodecyl benzene sulphonate, dioctyl sodium sulphosuccinate or sodium lauryl sulphate, which are known to stabilize radical cations, such as oxidised o-tolidine. Wetting agents are added to the impregnation solution 0.5-5%, preferably 1-3% -

To prepare the test papers according to the invention, an absorbent carrier, such as filter paper, cellulose or an active web, is impregnated with reagent solutions in volatile solvents. This is conveniently done in two or three separate steps. Thus, conveniently, the solution is first impregnated with a solution containing hydroperoxide, a wetting agent, a buffer and, if appropriate, a thickening agent. It is then saturated with a solution of the indicator and with a solution of the activator of the general formula I.

After drying, the test strips according to the invention are cut into smaller strips and are preferably arranged between the artificial foil and the fine-mesh net in accordance with German Patent Publication No. 2,118,455.

In order to detect peroxidatively active substances in the faeces, it is also possible to adapt the activators according to the invention to their reagent in an anhydrous membrane in accordance with German Patent Publication No. 1,598,153. This has the advantage that the surface of the test strip can be cleaned to detect a color reaction by simply wiping.

The following examples further illustrate the invention:

Example 1

The filter paper is successively saturated with the following solutions and dried at 40 ° C:

Solution 1 in 1.2 molar citrate buffer, pH 5 → 25 35.0 ml

Ethylenediaminetetraacetic acid, disodium salt 0.1 g

Dodecylbenzenesulfonic acid sodium salt 0.2 g 2,5-dimethylhexane-2,5-dihydroperoxide (from about $ 70) 1.6 g

Posforic acid trimorpholide 12.7 g

Ethanol 30.0 ml

Distilled water ad 100.0 ml 6 57025

A solution of 2-tolidine in 0.3 g of α-stilbazole 0.2 g

Toluene 100.0 ml

A white test paper is obtained which stains blue-green within about 5 to 20 seconds when dipped in blood-red urine. If the erythrocytes are intact, the papers are blue-green spotted. If hemolysis has occurred or if there is pre-existing free hemoglobin in the urine, the papers will turn solid blue-green. The sensitivity is about 5 erythrocytes / mm 2 or an equivalent amount of hemoglobin. A lower number of whole erythrocytes may, under certain conditions, cause discrete green spots on the test paper. Sensitivity to myoglobin corresponds to sensitivity to hemoglobin.

Example 2

The filter paper is saturated with the following solutions and dried at lämpöt0 ° C.

Solution 1 in 1.2 molar citrate buffer, pH 5.25 40.0 ml

Ethylenediaminetetraacetic acid, disodium salt 0.1 g

Water, dist. to 100.0 ml

Solution 2

Dioctyl sodium sulfosuccinate 0.5 g

Polyvinylpyrrolidone 0.5 g

3.5 g of diisopropylbenzene hydroperoxide (about 50%)

Phosphoric acid trimorpholide 12.7 g

Methanol is added in 100.0 ml

A solution of 3 o-tolidine 0.25 g

Compound I 0.25 g

Toluene 100.0 ml

Test papers are obtained which have practically the same properties as described in Example 1. The sensitivity is between about 5 and 20 Ery / mm · ^, although it decreases somewhat with the use of the next set of Compound I.

7 S 7 O 2 5

Compound I: Pyridyl-2-pyridyl-3-ethylene

Di- (pyridyl-2) ethylene 2- (4-methoxyphenylvinyl) pyridine 2- (4-methylphenylvinyl) pyridine Pyridyl-2-pyridyl-4-ethylene 2- (phenylvinyl) -4-methylpyridine

All of the compounds shown are known in the literature.