DE10030193A1 - Test kit for determining total protein and globulins, useful for diagnosing kidney disease, comprises copper sulfate as additional precipitant and polysaccharide - Google Patents

Abstract

A test kit for determining total protein (A) and globulins (B) in a biological fluid, comprising reagents that precipitate (A) and permit visual evaluation of the results of a precipitation reaction, in a solid formulation, apart from usual precipitants, copper sulfate as additional precipitant and at least one polysaccharide (II), is new. An Independent claim is also included for determination of (A), (B) and optionally hemoglobin (Hb) in biological fluid using the new test kit.

Description

The invention relates to a test kit for the determination of Total protein and globulins as well as hemoglobin, in biological fluids directly in a sample, especially in the urine. The test kit contains in addition to itself common reagents for total protein precipitation copper sulfate, at least one polysaccharide and optionally at least a monosaccharide and represents a reagent mixture in the form a granulate or in another solid formulation The invention also relates to a method for Proof of total protein and globulins as well as Hemoglobin in a biological fluid sample.

Methods for the qualitative and quantitative determination of Proteins (total protein) in urine, plasma, serum, liquor and in other biological fluids are numerous known and diagnostically relevant. The proteins can get through Precipitation and color reactions can be detected. Such Methods are used to detect numerous diseases. Of the Proof of proteinuria (protein excretion in the urine) is used especially for diagnosing various kidney diseases.

The protein detection can be qualitative and semi-quantitative with Test strips are made, usually a reagent which forms a color reaction with the protein. These Test strips have the disadvantage that they are exposed to light, Moisture and high temperature sensitive and unstable, i.e. not storable, whereby a Evaluation is no longer possible after longer periods of time. They also have insufficient analytical skills Sensitivity.

Other qualitative, semi-quantitative or quantitative Evidence is provided e.g. B. by adding sulfosalicylic acid to the urine or with the biuret reaction after precipitation with Trichloroacetic acid or perchloric acid, if necessary under Color additives. Known precipitation reagents are alongside Acids and others also heavy metal salts, acetone, alcohol, Ammonium salts and the like.

A test system for determining protein in the urine is in the Russian patent RU 2 077 060 C1. This Test system that is used to precipitate the protein with a reagent, possibly with a component that is insoluble in water is connected, is used, contains the reagent for Protein precipitation as a dense, solid granulate with a specific weight greater than 1.

However, the known test systems all have the disadvantage that you can only determine the total protein content. the Determination of the total protein content again does not allow a clear diagnosis nor the determination of the respective Severity of the disease. But this is for differential Diagnostics so z. B. of different types of Kidney diseases, leukemia, myeloma but also with others Illnesses important. So play alongside the total protein content especially the globulins and others. in determining the Kidney selectivity plays an important role. Evidence too of hemoglobin in the urine is present in the presence of hematuria as a result of damage to the urinary tract e.g. B. by kidney or Bladder tumors as well as other kidney diseases are an important one Indicator. For further diagnostic information, the individual proteins in the total protein (e.g. by means of Electrophoresis according to their molecular weight) be separated. Another disadvantage of known Test systems is an inconvenient and inconvenient for laboratory technicians dangerous manual labor, because any skin contact with the Reagents should be avoided. Furthermore you can Opacities arise that worsen the analysis result.

The object of the invention was therefore to provide a simple to find and deploy the applicable test kit of it permitted without major technical effort, in addition to the Total protein, the globulins and possibly hemoglobin, as additional evaluate diagnostic parameters.

According to the invention, the object is achieved by a test kit, of a reagent mixture in a solid, dense formulation represents the total protein and globulins with which Hemoglobin will be precipitated and the will continue to be a visual Ensures assessment of the result of the precipitation reaction, this solid reagent mixture becoming fixed or loose; z. B. in granulate or tablet form, in a transparent Test reagent container, such as B. a test tube, one Titer plate or the like. When using a This granulate has dense, solid granules specific weight greater than 1.

The test kit according to the invention contains in addition to the usual ones Precipitation reagents, as a further precipitation reagent copper sulfate and in addition at least one polysaccharide, whereby especially the total protein content and the globulin content is determined. In a preferred embodiment of the Invention, the reagent mixture also contains at least a monosaccharide, which makes a difference between various protein precipitates, e.g. B. Immunoglobulin and Can reach and determine hemoglobin precipitates.

According to the invention, the test kit contains the reagents the following% by weight:

Precipitation reagent (with copper sulfate) 85-95% by weight Polysaccharide 5-15% by weight

If the test kit also contains a monosaccharide, then it has to the reagent mixture according to the invention has the following% by weight:

Precipitation reagent (with copper sulfate) 70-90% by weight Polysaccharide 5-15% by weight Monosaccharide 5-15% by weight.

A mixture of one is used as the preferred precipitation reagent solid organic acid and an inorganic salt in Combination with copper sulfate.

The precipitation reagent is preferably in a mixture of the the following components:

Sulfosalicylic acid 50-95% by weight Sodium chloride 4-15% by weight Copper sulfate 1- 5% by weight

A soluble dextran is preferred as the polysaccharide contain. The preferred monosaccharide is glucose; especially D-glucose is used.

The test kit is supplied as a solid formulation, e.g. B. as granules, provided. To carry out the precipitation reactions is the solid reagent mixture (immobilized or loose) in a clear test reagent container, e.g. B. in a test tube, plastic bottle or in one Titer plate. The solid formulations, e.g. B. the Granules do not have a harmonious geometric shape. So can e.g. B. the granules and others. in the form of balls, chopsticks, Cylinders, lenses or the like are present. In a preferred As a variant, the test kit is a granulate in the form of a lens, where for the lenses a diameter of 2-8 mm and one Thickness of 1-4 mm is preferred. A lens shape favors z. B. a good solubility of the granules in one too investigating biological fluid.

The reagent mixture of the test kit is known per se Process for granulating or tableting chemical Mixtures made. So z. B. a saturated solution a precipitation reagent, which also contains the polysaccharide and Copper sulfate and possibly the monosaccharide contains, on one smooth plastic surface or in a clear one Test reagent vial added dropwise and dried out at 10 to 20 ° C.

In order to produce the lens shape of the granulate, an o. G. Solution on a plastic plate with wells that have a Have the desired lens shape, dripped and dried out.

The solid formulation produced in this way can be sealed in the test reagent container in which it is immobilized, or loosely in another closed glass or plastic bottle can be kept for up to 3 years.

The invention also relates to a method for detection of total protein and globulins and possibly of hemoglobin in biological fluids directly in a sample, especially in the urine.

In one embodiment, the method is for Protein determination characterized in that one has the reagent mixture according to the invention using Copper sulfate and polysaccharide e.g. B. in the form of granules into the biological fluid to be tested, preferably in Urine, - 0.5-1 ml of liquid in a test tube - gives and after a short exposure time of a maximum of 10 minutes total protein content based on the resulting precipitation, that is, based on the resulting white protein coating over the granulate, for sure.

Then the granulate is in the to be examined biological fluid homogeneously distributed. If necessary, the Mixture of granules and biological fluid mechanically treated, e.g. B. stirred or shaken until the granules dissolved in the liquid to be examined.

This homogeneous solution is then left for at least 30 minutes Stand still for 1.5 hours. In the presence of globulins after this time there will already be a in the solution Flake formation, within a short time after granulate dissolution, Any globulins that may be present then form a precipitate (Sediment deposition), which is semiquantitative with a photo or computer scan scale can be rated (Figure A).

The composition according to the invention enables surprisingly the determination of both the total protein and the Globulins in immediate succession in a sample of the to investigating biological fluid. The inventive Test kit allows you to find out the differences between the Sediment types of total protein and globulin precipitation to recognize and in a second step also the Determine the globulin content.

Another preferred method for protein determination, for Determination of globulins and for hemoglobin determination is that one z. B. 0.5 ml of the test biological fluid, preferably urine, into a Test tube with the solid according to the invention Reagent mixture, which in addition to the copper sulfate both a It contains polysaccharide as well as a monosaccharide, pouring it Let stand for 5-10 minutes, and the whole Protein content based on the resulting precipitate (white Protein ring above the reagent).

The total protein content can be determined semi-quantitatively with a photo scale, see Fig. 1. If a second protein ring (above the first) appears, see Fig. 2, this is also an indication that there is an increased alpha-1 microglobulin content .

The sample with its total protein content (of 300 mg / l) is then left to stand for at least another 30 minutes to 1.5 hours. If immunoglobulins are present, the protein ring changes into the so-called "mountain shape" after this time, ie the immunoglobulin precipitate partially emerges from the entire protein precipitate. If necessary, 0.1 ml of acetone is added to the sample to accelerate the immunoglobulin precipitation reaction to 20-50 minutes ( Fig. 3). The acetone can be added both at the beginning of the precipitation and after the total protein precipitation, whereby damage to the protein rings is to be avoided.

If there is also hemoglobin (approx. 300 μl / 1 l blood) in the sample to be tested, a red ring is visible over the reagent mixture according to the invention at the same time. This can also be clearly observed in the 30 minutes to 1.5 hours of the resting phase ( Fig. 5).

The composition of the test kit according to the invention makes it possible surprisingly the determination of both total protein and of globulins and hemoglobin in immediate succession only one sample of the biological to be examined Liquid.

The test kit according to the invention enables the Differences between the sediment types of total protein and from other protein precipitates (alfa-1- Microglobulin, immunoglobulin and also hemoglobin) recognize.

The test kit is particularly useful for in vitro diagnostics in medical (e.g. emergency doctor service) and veterinary Facilities and laboratories can be used and for Suitable for health diagnostics. It's easy to use, there the reagents are in a complete system. Included the test will only take a few seconds of manual labor feasible (approx. 5 seconds).

With the test kit according to the invention, only one single biological sample of both total protein can be detected as well as globulins and possibly hemoglobin in Combination with other proteins. So that is test kit according to the invention for first differential diagnostic purposes, especially in the case of kidney diseases, applicable, especially in the first step - the Determination of kidney selectivity. Furthermore, he can also serve to control the course of the disease.

The verification procedure can be carried out semi-quantitatively, with Photo scale or scanner scale can make the results objective be rated. The analysis result (a Protein sediment image) can either be obtained by means of a photograph (e.g. created with normal and digital photo equipment) or also directly from the test tube via the computer network Submitted to professionals for competent assessment. Subsequently, the invention is based on exemplary embodiments explained in more detail.

example 1

The test kit had the following composition:

Sulfosalicylic acid 80% by weight NaCl 7% by weight Copper sulfate 4% by weight Dextran 9% by weight

a) Comparative protein determination with pure protein solutions

Human serum albumin and γ-globulin were dissolved in normal urine at a concentration of 0.3 g / l. Three samples were compared:
Sample 1: albumin solution
Sample 2: globulin solution
Sample 3: albumin / globulin mixture (1: 1).

The test kit in the form of a Given granules. This was followed by the analytical procedure carried out as described.

Result

• 1. In all three samples were after 5 to 10 minutes Seeing protein rings above the granules, d. H. in each Protein is present in the sample.
• 2. The granules were shaken in all samples solved, in samples 2 and 3 one could after 30 resp. detect a clear flocculation after 45 minutes, after A typical sediment was at the bottom for 3 hours Record test tube. Sample 1 in which no globulin was contained, shows only a homogeneous turbidity. These results were scanned by computer (Figure B).

b) Determination of protein in human blood serum solutions

The test kit according to the invention was given in granulate form in each case to 6 serum and urine samples and in comparison therewith to 6 urine samples with pure albumin. They had a protein content of 0.02 to 0.8 g / l:
Serum + urine samples with 0.02 g / l, 0.05 g / l, 0.1 g / l, 0.25 g / l, 0.5 g / l, 0.8 g / l protein content in the mixture and
Urine samples containing 0.02 g / l, 0.05 g / l, 0.1 g / l, 0.25 g / l, 0.5 g / l, 0.8 g / l pure albumin.

The determination of protein and globulins was carried out as under a) described carried out.

Result

• 1. After 5 to 10 minutes there were protein rings in all samples seen above the granules, d. H. Protein was in everyone Samples available.
• 2. In four serum samples, clear flakes were 1 hour after Shaking can be seen in two samples (0.5 g / l and 0.8 g / l settled after 2 hours and in another 2 samples (0.1 g / l and 0.25 g / l) a typical sediment after 4 hours in the test tube, d. H. all serum globulins (α, β, γ) were found in the sample. The The size of the sediment corresponds to the level of the protein content.
• 3. The albumin samples were all only homogeneous Cloudy.

c) Determination of protein in urine samples from patients

The urine samples from 3 diabetics, from 2 patients with leukemia and 11 patients with kidney disease examined. The test kit was used in all of these samples Granulate form added.

Results

• 1. Typical protein deposits were found in all urine samples found over the granules, d. H. Total protein was available.
• 2. After making a homogeneous solution, the Urine sample from a leukemia patient and from 3 Patients with kidney disease first after 30-60 Minutes significant flakes and after 3 hours one typical sediment, d. H. there were globulins in these samples available.

Electrophoretic and immunological methods were used these results are confirmed.

Example 2

The test kit had the following composition:

Sulfosalicylic acid 75% by weight NaCl 4% by weight Copper sulfate 1% by weight Dextran 12% by weight D-glucose 8% by weight

a) Comparative protein tests of urine samples with Total protein (GE) and increased alfa-1 microglobulin content (a-mG)

50 urine samples with a certain protein content (from one medical laboratory) were delivered with the Test kit according to the invention analyzed in which the samples in a test tube was transferred, in which the test kit in a solid, fixed form with the above. composition found.

Result

• 1. Protein rings were found in all 50 samples after 5 to 10 minutes above the reagent mixture to see. H. GE is present in every sample.
• 2. In 14 cases there were no individual protein rings, but two rings had formed over the reagent mixture, see Fig. 2. In most cases these urine samples had an increased a-mG, ie an increased ratio of a-mG ( mg) to GE (1 g). The tendency - direct dependence of the ratio of α-mG / GE and an increased occurrence of protein images with two rings - was confirmed by means of statistical analysis.

Table 1

b) Comparative protein determination of pure protein solutions

Bovine serum albumin was found in normal urine with a Concentration of 2500 mg / l and each 0.5 ml were dissolved placed in two test tubes. In two others Test tubes were each 0.5 ml of a mixed solution (Albumin with the same concentration and gamma-globulin with given a concentration of 100n mg / l and as under 2a) tested, with an additional 0.1 ml of acetone was added.

Result

• 1. In all four samples were after 5 to 10 minutes Seeing protein rings above the reagent, d. H. in every sample is Protein present.
• 2. In the samples that additionally contained gamma-globulin, the protein ring turned into a so-called "mountain ring" after 40 minutes, ie the gamma-globulin precipitate partially emerged from the entire protein precipitate ( Fig. 3).

c) Comparative protein tests of urine samples with increased immunoglobulin content

There were 3 urine samples with a fixed, elevated one Immunoglobulin content (with the concentrations of 115 mg / l, 260 mg / l, 290 mg / l), a urine sample with little immunoglobulin (27 mg / l) and an unknown sample as under 2a).

Result

• 1. In all five samples were after 5 to 10 minutes Seeing protein rings above the reagent, d. H. in every sample there is protein present.
• 2. In the samples with a high immunoglobulin concentration and in the unknown sample, the so-called "mountain ring" was observed after 1.5 hours (as described under 2b), see Fig. 4.

d) Comparative protein studies of pure Albumin solution and albumin-hemoglobin mixtures as well as urine with microhematuria

A pure albumin solution (with a concentration of 1000 mg / l), an albumin hemoglobin mixture (approx. 300-600 µl Blood / l), as well as urine samples with and without microhematuria with the Test kit checked under 2a).

Result

• 1. In all samples with the albumin-hemoglobin mixture, a red hemoglobin ring was observed above the reagent (below the albumin) after 1 to 1.5 hours, see Fig. 5.
• 2. Similar results were obtained when analyzing Urine samples with microhematuria (approx. 300 Mkl blood / l urine) obtain.

Claims (13)

1. Test kit for the determination of total protein and globulins in biological fluids, the test kit containing reagents which precipitate the total protein and ensure a visual assessment of the result of the precipitation reaction, characterized in that it is in a solid formulation in addition to the usual precipitation reagents as a further precipitation reagent Having copper sulfate and additionally at least one polysaccharide. 2. Test kit according to claim 1, characterized, that in the solid formulation the precipitation reagents with 85-95 wt .-% and the polysaccharide are contained at 5-15% by weight. 3. Test kit according to claim 1 or 2, characterized in that the polysaccharide is a soluble dextran. 4. Test kit according to one of claims 1 to 3, characterized, that in the solid formulation for additional Determination of hemoglobin also includes at least one Monosaccharide is included. 5. Test kit according to claim 4, characterized, that in the solid formulation the precipitation reagents with 70-90% by weight, the polysaccharide at 5-15% by weight and the monosaccharide are contained at 5-15% by weight. 6. Test kit according to claim 4 or 5, characterized in that the monosaccharide is D-glucose. 7. Test kit according to one of claims 1 to 6, characterized in that the precipitation reagent is a mixture of a solid organic acid, an inorganic salt and Is copper sulfate. 8. Test kit according to claim 7, characterized in that the organic acid sulfosalicylic acid and that Inorganic salt sodium chloride is included in the mixture are present with copper sulfate in the following% by weight Sulfosalicylic acid 80-95% by weight Sodium chloride 4-15% by weight Copper sulfate 1-5% by weight. 9. Test kit according to one of claims 1 to 8, characterized in that it is present as granules, which has the shape of a lens and a preferred diameter of 2-8 mm and a thickness of 1-4 mm. 10. Test kit according to one of claims 1 to 9, characterized in that it may be immobilized in a test reagent vessel is located, such as B. in a test tube or Plastic bottle. 11. Procedure for the detection of total protein, globulins and possibly of hemoglobin in biological fluids directly in a sample using a test kit according to one of claims 1 to 10, characterized in that one the biological fluid to be tested with the test kit brings in contact and after an exposure time of a maximum of 10 minutes based on existing protein rings evaluates, based on the resulting Protein precipitate over the test kit of the Total protein content and if there is a second one Protein ring, an increased alfa-1 microglobulin content is determined, then the test kit is left for another 30 minutes to 1.5 hours on the biological Liquid act and then determined on the basis of itself changing the precipitate pattern globulins and hemoglobin. 12. The method according to claim 11, characterized in that one to accelerate the immunoglobulin precipitation reaction before or after determining the total protein content Add acetone to the sample.