GB1560077A

GB1560077A - Test composition for the detection or peroxidatively active substances

Info

Publication number: GB1560077A
Application number: GB3210476A
Authority: GB
Original assignee: Behringwerke AG
Current assignee: Siemens Healthcare Diagnostics GmbH Germany
Priority date: 1975-08-06
Filing date: 1976-08-02
Publication date: 1980-01-30
Also published as: BE844973A; NL7608492A; IT1065244B; JPS5221892A; FR2320553A1

Description

(54) TEST COMPOSITION FOR THE DETECTION OF PEROXIDATIVELY ACTIVE SUBSTANCES (71) We, BEHRINGWERKE AKTIENGESELLSCHAFT, a body corporate organised according to the laws of the Federal Republic of Germany, of Marburg/hahn, Federal Republic of Germany, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: The present invention provides a test composition for the detection of peroxidatively active substances, for example occult blood in biological fluids, especially in body fluids.

The detection of occult blood (invisible to the eye) in urine, faeces or vomitus is of great importance for the diagnosis of gastro-intestinal diseases or of diseases of the urinary tract and the kidneys. When small quantities of blood are detected in time a deterioration of the disease may be avoided by beginning treatment early enough.

The detection of occult blood has been used for a long time in medical diagnostics. For this purpose use is made of the peroxidative effect of erythrocytes or of hemoglobin. Originally benzidine was used as the chromogen which was oxidized by H202 in the presence of blood with the formation of a blue compound. The sensitivity of this reaction was improved in the course of time by the addition of quinoline (cf. Zeitschrift fir gereichtliche Medizin 12, (1928), page 216).

Because of the great importance of the detection of occult blood in screening examinations rapid test devices have been developed in recent years for this purpose. These devices generally comprise an absorbent carrier containing all the chemicals required for the reaction. As oxidants there are mostly used organic hydroperoxides, and as indicators o-tolidine or other chromogenic materials which are in most cases less dangerous to health than o-tolidine, for example, certain aromatic hydrazones (cf. US Patent Specification No.

3,558,435), pyridine derivatives (cf. US Patent Specification No. 3,630,847), hydrazine derivatives (cf. British Patent Specification No. 1,215,778) and other aromatic azines (cf. US Patent Specification No. 3,627,697).

Test papers prepared with the use of such chromogenic materials, however, have insufficient sensitivity and must be sensitized with complicated substances to improve the sensitivity, for example, with quinine (c.f. US Patent Specification No. 3,290,117) or with certain benzoquinolines (cf. US Patent Specification No. 3,853,472).

3,3',5,5'-Tetramethylbenzidine is a non-carcinogenic substance suitable for the detection of blood (cf. Tetrahedron, volume 30, page 3299). In the cited passage the use of the above compound is limited to a test in solution. This however, has the disadvantage that the colour complex formed is stable for a short period only.

The present invention is based on the observation that this indicator and its acid addition salts are suitable for the preparation of test strips.

The invention accordingly provides a test strip for the detection of a peroxidatively active substance, which comprises a carrier strip impregnated with a peroxide, 3,3',5,5'd tetramethylbenzidine or an acid addition salt thereof, and quinoline or isoquinoline.

When the test strip is contacted with a peroxidatively active substance, the colour complex formed is surprisingly stable for a substantially longer time than it is in solution. The test strip moreover shows very sensitive results. The formation of the color complex is further accelerated by the addition of quinoline, a quinoline derivative, or isoquinoline. The sensitivity of such a test strip is ten times superior to that of a test strip based on o-tolidine.

One or several peroxides are present, preferably organic hydroperoxide, for example, 2,5-dimethylhexanedihydroperoxide. The test strip may, moreover, comprise one or more auxiliaries, for example, wetting agents, thickening agents, buffer substances and/or stabilizers.

The individual chemicals may be applied to the absorbent carrier strip together in a dissolved state preferably in a readily volatile solvent or, when required by special circumstances, in separate solutions.

To carry out a test using a test strip of the invention the test strip is contacted for a short time with the liquid to be examined, for example, a body fluid. After only a few seconds the presence of peroxidatively active substances may be determined semi-quantitatively by means of the change in colour or optionally by comparison with a colour chart. This test is part of the invention.

The following Example illustrates the invention: EXAMPLE: Filter paper No. 2316 of Messrs. Schleicher and Schiill was impregnated with solution 1 and dried for 1 hour at 40"C, and was then impregnated with solution 2 and was again dried for 1 hour at 40"C.

Solution 1: 1.2 molar citrate buffer, pH 5.0 35.0 ml 2,5-dimethylhexanedihydroperoxide 1.6 g phosphoric acid trimopholide 12.7 g ethanol 30.0 ml deionised water 35.0 ml Solution 2: 3,3', 5,5'-tetramethylbenzidinedihydrochloride 0.3 g quinoline or isoquinoline 0.2 g methanol 100.0 ml A test paper prepared in said manner indicated by a blue coloration a content of blood from only 0.1 mg per 100 ml of solution to be examined. A similar paper containing o-tolidine instead of the tetramethylbenzidine was only capable of indicating a content of blood from 1 mg per 100 ml of solution to be examined.

Individual erythrocytes were indicated by blue points, whereas haemoglobin produced uniform blue coloration. Myoglobin, which may occur in urine, for example after a myocardial infarction, was also indicated by a uniform blue coloration.

A test paper prepared according to Example 1 and using the tetramethylbenzimidinehydrochloride exhibited an unchanged coloration even 40 hours after having been dipped into the solution to be examined, whereas in the same test carried out using an analogous liquid reagent the color complex remained stable only for about 2 hours.

When o-tolidine was used as the chromogenic agent instead of the tetramethylbenzidine, the colour complex formed on the test strip remained stable for 10 hours, and the colour complex formed in solution remained stable for about 1 hour.

WHAT WE CLAIM IS; 1. A test strip for the detection of a peroxidatively active substance, which comprises a carrier strip impregnated with a perioxide, 3,3', 5,5'-tetramethylbenzidine or an acid addition salt thereof, and quinoline or isoquinoline.

2. A test strip as claimed in claim 1, wherein the peroxide is an organic hydroperoxide.

3. A test strip as claimed in claim 2, wherein the peroxide is 2,5dimethylhexanedihydroperoxide.

4. A test strip as claimed in any one of claims 1 to 3, wherein the carrier is impregnated with one or more auxiliaries.

5. A test strip as claimed in claim 4, wherein the auxiliary or auxiliaries is or are selected from wetting agents, thickening agents, buffers and stabilizers.

6. A test strip as claimed in claim 1, and which is substantially as described in the Example herein.

7. A method for determining a peroxidatively active substance, which comprises contacting a solution or suspension comprising the substance with a test strip as claimed in any one of claims 1 to 6 and observing the resulting colour of the strip.

8. A method as claimed in claim 7, wherein the solution or suspension is a body fluid.

9. A method as claimed in claim 8 or claim 9, wherein the peroxidatively active substance is blood.

10. A method as claimed in claim 7, carried out substantially as described in the Example

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **. 2,5-dimethylhexanedihydroperoxide. The test strip may, moreover, comprise one or more auxiliaries, for example, wetting agents, thickening agents, buffer substances and/or stabilizers. The individual chemicals may be applied to the absorbent carrier strip together in a dissolved state preferably in a readily volatile solvent or, when required by special circumstances, in separate solutions. To carry out a test using a test strip of the invention the test strip is contacted for a short time with the liquid to be examined, for example, a body fluid. After only a few seconds the presence of peroxidatively active substances may be determined semi-quantitatively by means of the change in colour or optionally by comparison with a colour chart. This test is part of the invention. The following Example illustrates the invention: EXAMPLE: Filter paper No. 2316 of Messrs. Schleicher and Schiill was impregnated with solution 1 and dried for 1 hour at 40"C, and was then impregnated with solution 2 and was again dried for 1 hour at 40"C. Solution 1: 1.2 molar citrate buffer, pH 5.0 35.0 ml 2,5-dimethylhexanedihydroperoxide 1.6 g phosphoric acid trimopholide 12.7 g ethanol 30.0 ml deionised water 35.0 ml Solution 2: 3,3', 5,5'-tetramethylbenzidinedihydrochloride 0.3 g quinoline or isoquinoline 0.2 g methanol 100.0 ml A test paper prepared in said manner indicated by a blue coloration a content of blood from only 0.1 mg per 100 ml of solution to be examined. A similar paper containing o-tolidine instead of the tetramethylbenzidine was only capable of indicating a content of blood from 1 mg per 100 ml of solution to be examined. Individual erythrocytes were indicated by blue points, whereas haemoglobin produced uniform blue coloration. Myoglobin, which may occur in urine, for example after a myocardial infarction, was also indicated by a uniform blue coloration. A test paper prepared according to Example 1 and using the tetramethylbenzimidinehydrochloride exhibited an unchanged coloration even 40 hours after having been dipped into the solution to be examined, whereas in the same test carried out using an analogous liquid reagent the color complex remained stable only for about 2 hours. When o-tolidine was used as the chromogenic agent instead of the tetramethylbenzidine, the colour complex formed on the test strip remained stable for 10 hours, and the colour complex formed in solution remained stable for about 1 hour. WHAT WE CLAIM IS;

1. A test strip for the detection of a peroxidatively active substance, which comprises a carrier strip impregnated with a perioxide, 3,3', 5,5'-tetramethylbenzidine or an acid addition salt thereof, and quinoline or isoquinoline.