DE1498601A1 - Diagnostic agent for the detection of components of whole blood - Google Patents

Description

GmbH 13o1

Mannhe im

Dr. Expl.

Diagnostic agent for the detection of ingredients of the rabid blood

The detection of ingredients in whole blood has ceased so far very cumbersome, complex and time-consuming process, which only in appropriately equipped laboratories and with trained Qualified personnel can be carried out. First of all, the serum or plasma must be obtained from the whole blood, in which only then can the substances of interest be determined; otherwise the strong coloring property of the Hemoglobins interfere with all color reactions “There have also been processes recently have been described, which enable the detection of glucose or urea in whole blood in a simplified way (USA patent 3,092,465, British Patent 922,665). The principle of this new method consists in that a test paper strip is coated from the outside with a semipermeable wall} you put on one Such strips of whole blood will enter the serum with the ingredients through the semi-permeable film and leads to a color reaction in the paper, while the red blood cells remain on the film. After a certain time, the red blood clot must be washed off, so that you can see the reaction color in the paper. This method is certainly a simplification compared to the laboratory methods described above, but it still has significant disadvantages: In order to be able to assess the reaction color, the red blood components must be wiped off or washed off; if this If it does not happen properly, it can lead to incorrect assessments. The production of these test strips is also relatively laborious and laborious.

■ A quick determination of substances contained in the blood inexperienced persons as a quick examination method at the bedside, in the event of accidents or as a "screening test" in clinics is of great importance for many substances.

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^ · Glucose j The determination of the glucose concentration in the blood is particularly important in diabetic patients, for example in accidents where it must be determined quickly whether a coma is present as a result of eyperglycaemia or hypoglycaemia. The monitoring of blood glucose levels when preparing a patient for appropriate medication is also of interest. When finding latent cases of diabetes, a simple and quickly practicable method for determining G-glucose in I-full blood (“screening test”) is important.

2. Urea t The detection of the urea content in the blood is important for the assessment of animal diseases and for the detection of undetected azotaemias.

3. serum cholinesterase ; A simple test to check serum cholinesterase is required to assess various liver diseases as well as in cases of poisoning by pesticides.

4 x phenylalanine ; In order to be able to determine whether an oligophrenia phenylpyruvica is present in the infants' first days of life, the delivery wards would have to have a simple "screening test" for the phenylalanine content in the blood. So far, one has been dependent on detecting phenylpyruvic acid in the urine - a method that only leads to reliable results from the 6th week of life. The subsequent registration of the infants is, however, extremely cumbersome; a lot of time is also lost here, so that it is often no longer possible to successfully treat this illness leading to dementia in good time.

5 » galactose ; The early identification of infants with galactosaomy by a rapid determination of the galactose in the blood is of similar importance.

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Other substances whose rapid determination in the blood is important are ζ.Β * Phosphatesen, transaminases, dehydrogenases, trypsin, Histidine, uric acid.

For the detection of glucose in the blood, the specific action described by W. Pranke and P. Lorenz [Idebigs Annalen 5} 2 , (1937) »pp. 1-28] is usually used; 33s this involves the oxidation of glucose Gluconic acid, which is carried out by means of molecular oxygen in the presence of glucose oxidase and a "buffer" at pH 5. The released hydrogen peroxide oxidizes an Eedox indicator in the presence of peroxidase. P-phenylenediamine and those known under the name "Hadi" reagents can be used as indicators become mixtures of phenols and aromatic amines, especially substances with Benzidinstruktur, for example, the o-folidin. In this Glucoseifachweis to obtain a more or less strong blue staining according to the Glueosekonzcntrationj by the addition of yellow dyes can be achieved corresponding Griintöne. impregnated to an absorbent body , for example filter paper, with the mentioned Heagenzien, so one obtains a yellow paper that is colored green to a greater or lesser extent by glucose solutions. With such a paper, glucose can be detected qualitatively and semi-quantitatively in liquids of little color, for example urine. ! Hisses to this paper, however, in blood or smeared it with blood, although discoloration occurs depending on the glucose levels in the blood. However, a green coloration cannot be clearly identified » let alone gradations in the color intensities; a sufficient chromatography effect due to the spreading of the blood fluid in the eye-capable carrier cannot be observed either.

One tries in such testing with whole blood, an additional chromatographic Auftrennng of blood in the absorbent solid support by known coagulation-demanding means trnd order to reach a determination of the disturbing blood cells, we get to our findings with numerous substances no or very inadequate Heeultat. Proved to be unusable »see liua- s

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Thrombin preparations, adrenochrome monosemicarbazone, spinal cord extracts, 2-methyl-1 ^ -naphthoquinone derivatives, uranyl acetate, calcium chloride, Calcium gluconate. Macromolecular substances did not show any either useful or sufficient effects ι Carrageanate, pectins, Polygalacturonic acid derivatives, vinylpyrrolidone-vinyl acetate copolymers, Polyvinyl alcohols, polyethylene glycols, polyacrylamides, gelatine, polyglycol 2o ooo,

Surprisingly, it has now been found that by adding Amino acids or amino acid amides or their salts test papers are given, which when immersed in blood, a strong separation of result in red blood substance and only black-colored blood fluid, «6« load the special color reaction in the wetting area Serum proportions is clearly visible. As aoino acids are special Glycocolla and alanine come in handy as amino acid amides E.g. * in question glycinamide, asparagine, glutamine.

If, for example, one thaws a glucose impregnated with the addition of glycine

Test paper in blood, so will the immersed part of the paper strip colored reddish brown by the blood. Liquid then rises from this strongly colored zone without any particular color of its own second zone is colored as a result of the glucose-skin reaction in 'Presence of a yellow dye more or less green depending on the glucose concentration in the blood. A similar one ^ The effect also occurs across the paper with one-sided ^ Wetting by the blood ι If the paper is thick enough, the color reaction can be clearly seen on the back.

From the German Auslegeschrift 1 153 92o is a diagnostic Means for the determination of ketone compounds in body fluids became known that from one with sodium nitroprusside with the addition an Aminoesaigsäu're impregnated, absorbent carrier. ., .. You aminoeaetic acid is used in this case for the purpose of achieving the necessary Sensitivity of the color reaction added (cf. USA patent 2 5o9 Ho). Such an increase in sensitivity is with the diagnostic gowns according to the invention are not required) the areapitture addition is only used here to separate the blood pigments ! · From the blood fluid needed to observe the color reaction well

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can. In addition, the German method is suitable Aüslegeschrift 1 153 92o only for the determination of ketone bodies in practically colorless liquids such as urine} the red-violet color reaction cannot be successfully tested in whole blood in this test watch.

To produce the diagnostic agent according to the present invention, the absorbent carriers, for example paper test strips, are impregnated in the usual way with the specific reagents and indicators, adding 1-2o% by weight of an amino acid or an amino acid amide or their salts to the impregnating solutions. Analogously, the part of the test strip soaked with the amino acid or the amino acid amide or its salts can also be separated from the coloring zone if the whole blood to be tested is applied to the amino acid-containing part and allowed to diffuse into the coloring part. The coloring zone and the precipitation zone can also be separated into two separate test strips that are in contact with one another.

A particularly useful embodiment of the invention diagnostic agent is obtained by sealing the test strips on transparent material in accordance with German utility model no. 1 852 316} the test combinations obtained in this way are good readable and only require a drop of whole blood. It has proven to be very beneficial if you have the actual test strips with one second, slightly wider strip of paper combined with one Solution of amino acids or amino acid amides or their salts is impregnated, and these two strips on top of each other on a plastic film that the wider strip from the outside seals the narrow one Evenly covers the test strip (see illustration). The test strip itself does not need in this case with amino acid or amino acid amide or to be soaked in their salts. To make these test combinations you seal the two paper strips on one side on a sheet of film and then cuts this web across, for example 5 "in width

Stripes. In this way, plastic strips are obtained at one end there is a test disc. located only from one side The other side, which is covered by the film, is absorbent

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a square teat plate that goes through from above and below appears enclosed by a color-contrasting stripe (see illustration). Put a drop of blood on the absorbent Part of the stripe, this is colored red by the blood; against it if the test platelet itself is not influenced by the color of the blood, it gives the specific color reaction of the blood to be detected Substance. Since the blood drop does not directly contact the test plate and comes into contact with the reagents, the test strip can briefly against the drop of blood emerging from the pinger or earlobe be placed. This embodiment of the diagnostic agent according to the invention therefore allows the simplest possible handling.

Principle all water-insoluble polymers in question as a transparent material, for example polyvinyl chloride, and polyterephthalic piyäthylenbeschiehtete carrier or pollen (eg Polyterephthalsäureesterfolie, acetate _r polypropylene, polyamide Super). Decisive for the selection of the suitable plastic is first and foremost its good weldability and a favorable price. Polyvinyl chloride films are preferably used because they are available in large thicknesses and are cost-effective. The sealing is carried out in a known manner either by the thermal impulse or thermal contact method or by means of high frequency (see German utility model Mr. 1 852 516).

The following examples show the preparation of the inventive diagnostic agent explained.

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Example 1 t production of a test strip suitable for the detection of glucose in whole blood.

Impregnation solution It

Peroxidaee ad o, o12 β Glucose oxidase 1.3 η Tartraein o, 1 Il prim * potassium phosphate o, 269 Il seo. Sodium phosphate o, oo4 Il Glycocolla 5.0 It acetone 1o, o ml least. water 1oo «o Il

Impregnation solution II "

o-tolidine 0.42 g

Acetone to 100.0 ml

With impregnation solution I and II, filter paper Schleicher & Bohtill Kr. 2} 16 impregnated, dried and in strips of 5 mm width cut *

If one dips such a strip into a drop of blood, e.g. on the finger or on the earlobe, about 3 nuh deep, it becomes over the immersed zone a zone of approximately the same width by a ■ fluid free of red blood pigment, depending on the moistened Öluoosegehalt is more or less strongly colored green. To this So I leave glucose in a concentration of less as 25 mg- $ clearly prove.

Example 2 «Glucose test strips, sealed onto plastic film.

Filter paper Schleicher & Sohüll No. 2316 is first treated with the impregnation solution I given in Example 1, but the addition of glycocolla is missing. It is then impregnated with the impregnating solution II given above, dried and the paper is cut into strips 5 μm wide.

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Another batch of filter paper Schleicher & SchÜll No. 2316 is made impregnated with a glycoll solution of the following composition

Glycocolla 5 g

least. Water ad 100 ml

and cut to the desired width of 9 mm.

The glycoll paper strips obtained in this way are placed over the glucose touch paper strips described above and passed on a heat-sealable polyvinyl chloride film through two rollers, one or both of which are heated. The combined papers are welded onto the foil through the thermal contact. This is then cut transversely into 5 mm wide strips. The latter represent the finished test strips (see figure) »

If the test strips are moistened with a drop of blood at the absorbent point, the yellow, square tea plate turns green to a greater or lesser degree in about 1-2 minutes, depending on the glucose content. The white paper, impregnated with glycocolla, is colored red-brown by the blood pigment. In this way, glucose can still be clearly detected in a concentration of less than 25 mg.

Example 3 Production of a test strip suitable for the detection of serum holinesterase in whole blood.

Impregnation solution Is

Butyrylehol iodide, 5 g

a-alanine 5 "

least. Water to 40 ml 1n sodium hydroxide solution (q.s. Ms ajtf pH 7, o)

Methanol 1o ^H

Impregnation solution Ut

Bromothymol blue o, 55 g

Methanol to 50.0 ml

BATH ORIGINAL

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Filter paper (Schleicher & Schüll No. 2516) is used one after the other treated with the impregnation solutions I and II and processed in the same way as in. Example 1 described.

The impregnation solution for the blood dye separation paper has the following Composition i

oc-alanine 5 g

least. Water ad 100 ml

Impregnation on filter paper Schleicher & Schüll No. 2516.

The cholinesterase test paper strips and the alanine paper strips are sealed and cut on foil according to example 2. The test strips obtained in this way are moistened at the absorbent point with a drop of blood, this is how the orange-colored test plate changes color initially blue-green and then again more or less quickly orange over the most varied shades of green. The speed of the color change is a measure of the serum cholinesterase activity in the Blood. If you compare the resulting color after a certain time With a color scale, the activity can be determined The brighter the test plate, the higher the serum cholinesterase activity, the smaller the change in color after the initial blue-green coloration the lower the activity.

Example 4 ^; Production of a test strip suitable for the detection of galactose in whole blood.

Impregnation solution I:

Peroxidase o, o12 g

Galactose oxidase 0.5 "

prim. Potassium phosphate, o.746 "

lake. Sodium phosphate 2.585 "

Acetone 10, o ml

least. Water ad 1oo, o "

Impregnation solution II:

o-tolidine 0.42 g

Acetone ad 100.0 ml ^!

_1o 9 0 9 8 U / 0

Filter paper (Schleicher & Schüll No. 2516) is successively with impregnated with the impregnation solutions I and II, dried and cut.

The impregnation solution for the blood dye separation paper has the following Composition:

Glycinamide hydrochloride 5 g least. Water ad 1ooml

Filter paper Schleicher & Schüll No. 2316 is impregnated with this solution.

The galactose test strips and the glycinamide paper strips will be Sealed and cut as described in Example 2 on plastic films. If the test strips obtained in this way are moistened on the absorbent area with a drop of blood, the square test disc turns blue to a greater or lesser extent depending on the galactose content. The rest of the paper is colored red-brown by the red blood pigment. So Galactose can still be found in a concentration of less than 7.5 mg- $ prove.

Example 5 ? Urea test papers.

Urease is mixed with a suitable buffer and a pH indicator applied to a filter paper. Such urea test papers can regarding the pH and the capacity of the buffer as well differ in the type of indicator. The indicator change is specific for very specific amounts of urea.

A test that works at urea concentrations below 50 mg-fo yellow-orange

and is colored red at concentrations above 50 mg- $, can be done by means of a

0.1 mol of phosphate buffer of pH 6.6 and phenol red as an indicator will.

Using an o, 2 mol phosphate buffer of pH 6.6 and phenol red as An indicator is obtained from a test on urea concentrations below $ 100 mg with a yellow-orange color and over $ 100 mg with a Red coloring reacts.

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_f -By use receives a 0.4 mol Phosphatpuffera pH of 5 '5 and phenol red as an indicator to a test that shows a yellow-orange coloration in amounts of urea of less than 2oo mg-fo and is colored red at higher amounts of urea.

These urea test papers are processed further into test strips in combination with the blood dye separation papers described in Examples 2, 3 and 4 according to the method given in Example 2. If the test strips obtained are moistened with a drop of blood at the absorbent point, the indicator color changes at the corresponding urea content in the square test plate. Depending on the type of buffer, amounts of urea can be determined in different concentration ranges} e.g. urea below 5 ° ws-fe * above 50 mg- $ but below 100 mg- $, above 100 mg-fo but less than 2oo mg- $, over 2oo mg- $.

Example 6 1 Phenylalanine test papers.

L-amino acid oxidase (from snake venom), peroxidase and o-tolidine are together with a 0.2 mol phosphate buffer with a pH of 6.5 a filter paper applied. A paper impregnated in this way is covered with a blood dye separation paper according to Example 2 Test strips processed. If you moisten these strips with L-phenylalanine containing agents Blood, this gives the test plate a blue color. Thus, children who are already in the first week of life can be Phenylketonuria.

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Claims (2)

Claims 1. Diagnostic means for the detection of constituents of whole blood, "consisting of an absorbent carrier, which is impregnated with specific reagents and indicators, characterized by the addition of amino acids or amino acid amides" or their salts, "which cause the whole blood to be separated into blood cells and practically colorless blood fluid, the specific test reaction taking place outside the red-colored area. 2. Diagnostic agent according to claim 1, characterized in that the amino acids or amino acid amides or their salts are applied to an absorbent carrier which is separate from the indicator carrier but is in contact with it. 3 · Diagnostic agent according to Claims 1 and 2, characterized in that the indicator carrier is covered on one side by the absorbent carrier which is impregnated with a solution of amino acids or amino acid amides or their salts, while on the other side it is covered by a transparent material is covered through which the color reaction of the test can be observed. SAD OFHGWAL 909814/070 $