IE45450B1 - Test strip - Google Patents

Abstract

1523581 Detection of amino acids KNOLL AG 28 June 1977 [12 Aug 1976] 26915/77 Heading G1B A test strip for detecting amino acids comprises an L-amino acid oxidase, a peroxidase and a chromogen, and comprises two layers, one layer containing one of these three reaction components and the other layer the other two reaction components. Examples relate to test strips comprising raw snake venom from Agkiotrodon rhodostoma (A.r.), peroxidase 4-amino-antipyrine 2, 4-dichlorophenol; snake venom from Bothrops atrox, peroxidase, 4-amino-antipyrine, 2, 4-dichlorophenol and as buffer 0À36M 2-(N-morpholino)- ethane sulfonic acid; snake venom from A.r., peroxidase and 2, 2'-azino-di [3-ethylbenzthiazoline sulfonic acid-(b)]-diammonium salt. Other chromogens are: o-diamioidine, o-toluidine and a mixture of 4-amino-2, 3-dimethyl-1-phenyl-3-pyrazolin-5- one (4-aminoantipyrine) and 2, 4-dichlorophenol.

Description

The present invention relates to a test strip for a rapid and simple determination for a variety of amino acids, particularly phenylalanine, in biological liquids.

The determination of phenylalanine is of signifi5 cance for the diagnosis of phenylketonuria. A number of methods for demonstrating this amino acid are already known [cf., for example, Hallnann, Klinische Chemie and Mikroskopie, 10th Edition, page 217, Thieme Verlag (1967) Merck, Klinisches Labor, 12th Edition, page 1OO, Merck IO Darmstadt (1974)]. However, these tests are either very unspecific or very complicated and, thus, are unsuitable for routine examinations in a physician's practice.

It has now been found that a number of amino acids can be determined easily and rapidly with a test strip which contains an L-amino acid oxidase, a peroxidase, and a chromogen, said test strip containing two layers one on top of the other, one of which layers contains said Lamino acid oxidase, or said peroxidase, or said chromogen and the other of said layers contains the other two materials.

L-amino acid oxidases are components of the venom of snakes (e.g. Aqkistrodon piscivoris, Agkistrodon rhodostoma, Bothrops atrox. Echis carinatus, and Crotalus addmanteus). Such enzymes are also present in the liver of the rat [cf. Barman, Enzyme Handbook, Volume I, pages 176—177, Springer Verlag (1969)]. - 3 The L-amino acid oxidase from Aakistrodon rhodoatoma (the Malayan pit viper) can be prepared according to U.S. patent 3,657,416, incorporated herein by reference. The«enzyme ia obtained in fraction 7 (cf. column 3, lines 6Ο~γ65 of the patent). Instead of the purified enzyme, the crude snake venom can also be used, since the accompanying materials which are present therein do not disturb the test.

Any of thre known peroxidases can be employed in the test strip. Such materials are disclosed, for instance, in The Enzymes, Vol. 8, Academic Press (1963), pages 227—274, incorporated herein by reference. As a peroxidase, the peroxidase obtained from horseradish is particularly suitable.

As chromogens or chromogenic systems (DH^), any hydrogen donor having a stable color after oxidation can be employed. Exemplary materials include o-dianisidine; o-toluidine; 2,2'-azino-di-[3-ethylbenzthiazoline sulfonic acid-(6)]-diammonium salt; and a mixture of 4-amino-2,320 dimethyl-l-phenyl-3-pyrazolin-5-one (4-aminoantipyrine) and 2,4-dichlorophenol.

For preparation of the test strip, the L-amino acid oxidase, peroxidase,and the chromogen are applied to an absorbent solid substrate, preferably filter paper. Soft absorbent filter papers are particularly suitable.

If the test strip is brought into contact with a solution which contains phenylalanine, for example, the following reactions occur; CgH5—CH2—CH—COOH L-amino acid oxidase » NH, C&H5—CH2—CO—COOH + NH3 + H2O2 Peroxidase D + 2H2O The L-a-aino acid oxidase transforms phenylalanine at a pH valu- of 7.5—10 into the corresponding a-ketocarboxylic acid. In this reaction, hydrogen peroxide is generated, which, in the presence of a peroxidase, forms the dye (D) from the chromogen or chromogenic system (dh2) The concentration of the reactants in the test strip can be so chosen that a visible reaction only occurs if the test strip is brought into contact with a body fluid whose content of amino acid is pathologically elevated.

Phenylalanine reacts with the aforementioned Lamino acid oxidase. In addition, the enzymes oxidize, also within a short period of time, some other amino acids. Thus, for example, with the venom of Agkistrodon rhodostoma or Bothrops atrox, or the L-amino acid oxidase isolated therefrom, tyrosine and leucine can be oxidized and demonstrated with the test strip according to the present invention. This is very important for the diagnosis of cirrhosis of the liver.

A better understanding of the present invention and of its many advantages will be had by referring to the following specific examples given by way of illustration. >454 GO - 5 Example 1 The test strip comprises two layers which are prepared as follows: Layer No. 1: Filter paper (type 597 NF, prepared by Schleicher and Schull) is moistened with a saturated aqueous solution of 2,4-dichlorophenol and subsequently dried in a drying oven at 50°C. The process is repeated. Subsequently, the filter paper is moistened with a solution comprising one part of 4-aminoantipyrine solution (25 mmol/1) in 1/15 M phosphate buffer (pH == 8.0), one part of saturated aqueous 2,4-dichlorophenol solution, and one part of peroxidase solution, 100 mg/1) in 1/15 M phosphate buffer (pH = 8.0), and is then dried.

Layer No. 2: Filter paper (597 NF) is moistened with a solution containing lo g/1 of lyophilised raw snake venom from Agkistrodon rhodostoma (content of L-amino acid oxidase is about 10%) in 0.9% sodium chloride solution, and is subsequently dried at 60°C.

Layers No. 1 and No. 2 are placed one on top of the other and employed for testing. The test strip con2 tains about 10 mcg/1 of L-amino acid oxidase per cm and colours only if it is brought into contact with serum whose phenylalanine content is above normal.

Example 2 Example 1 is repeated but one layer contains the peroxidase and the snake venom and the other contains the 4-aminoantipyrine and the 2,4-dichlorophenol. The snake venom was isolated from Bothrops atrox. As a buffer, 0.36 M 2-(N-raorpholino)-ethane sulfonic acid buffer (pH » 9.6) was used.

Example 3 Example 1 is repeated except that 2,2'-azino-di[3-ethylbenzthiazolinesulfonic acid-(6)]-diammonium salt is used as the chromogen.

When the test outcome is positive, the test strips of Examples 1 and 2 turn to a rose to red colour. The strip prepared according to Example 3 becomes blue-green.

Claims (2)

1. CLAIMS:1. A test strip for detecting amino acids comprising an L-amino acid oxidase, a peroxidase and a chromogen, said test strip comprising two layers one on 5 top of the other, one of which layers contains either said L-amino acid oxidase, or said peroxidase, or said chromogen, and the other of said layers contains the other two materials.

2. A test strip for detecting amino acids substan10 tially as hereinbefore described and as illustrated in the foregoing Examples.