DE202011105872U1 - Test reagent for a rapid test for protein determination in biological fluids - Google Patents

Abstract

Test reagent for a rapid test for protein determination in biological fluids, especially in urine, comprising a reagent mixture and a reagent vessel, characterized in that the reagent mixture contains urea, citric acid, hydrochloric acid and sodium chloride in addition to a conventional dye and a solvent.

Description

The invention relates to a test reagent for a rapid test and a method for protein determination in biological fluids, in particular in urine, comprising a reagent mixture and a reagent vessel. According to the invention, the reagent mixture additionally contains urea, citric acid, hydrochloric acid and sodium chloride in addition to a customary dye and a solvent. The combination of dye, precipitating reagents and the other substances of the reagent mixture, which is located in a preferably closed reagent vessel, is designed so that the interaction of color reaction and turbidity reaction contributes to the formation of a colored protein product, which due to the special optical properties (type and intensity the color, as well as hues and possibly image of colored turbidity) is in clear dependence on a determined protein concentration in the liquid to be tested.

Methods for the determination of proteins (total protein) in urine, serum and other biological fluids are widely known and diagnostically relevant. The proteins may, for. B. be detected by precipitation reactions. Such methods are used to detect many diseases. The proof of proteinuria (protein excretion in the urine) is used before age for the diagnosis of various kidney diseases. Many qualitative or quantitative methods take place for. B. by the addition of sulfosalicylic acid to the urine or with the biuret reaction after precipitation with trichloroacetic acid or perchloric acid, if known Known precipitating reagents are in addition to acids u. a. also heavy metal salts, acetone, alcohol, ammonium salts and the like.

A test system for protein determination in urine is in the Russian patent RU 2 077 060 C1 described. This test system contains the reagent for protein precipitation as a dense, solid granulate with a specific gravity greater than 1. The known test systems (test granules) and its modifications ( DE 100 30 193 A1 and DE 101 44 436 A1 However, all have the disadvantages that this method need special lighting devices, because in daylight, the result is visually complicated to evaluate. The protein detection can be carried out qualitatively and semi-quantitatively with test strips, which usually contain a reagent that forms a color reaction with the protein. These test strips have the disadvantage that they are sensitive and overly sensitive by light, moisture and high temperatures. In addition, they have insufficient analytical sensitivity. They usually have uncomfortable, for evaluation too small (5 × 5 mm), test fields.

A protein determination with the dye Coomasi G-250 is also known. But this test only needs photometric results measurements (there is no way to visually semi-quantitative assessment) and this is not a quick test ( Bredford, Anal Biochem. 72, 1976 ).

However, all known test systems and test methods all have the disadvantage that one must evaluate with them an analysis of the analysis result at certain times, since it is not durable and assessable. For example, one can not simultaneously examine more than a single sample with known test strips. Thus, none of the known tests can be used as a screening test, especially in series of diagnostic measures.

The object of the invention was therefore to find a test for rapid and convenient protein determination in biological fluids and to provide such that it allows an evaluation without much technical effort, and is suitable for semi-quantitative, as well as quantitative assessments, and both as a single Test as well as for series screening analysis is suitable. The stained test analysis product must be visible and stable over time. Another task was a simple and convenient method for use o. G. Find and provide tests.

The test reagent according to the invention for a rapid test for protein determination in biological fluids, in particular in urine, is characterized in that the test reagent comprises a reagent mixture which, in addition to customary dyes and solvents, additionally contains urea, citric acid, hydrochloric acid and sodium chloride.

This combination of dye and precipitation reagents in a reagent mixture, which is preferably in a closed reagent vessel, is designed so that an interaction of color reaction and turbidity reaction to form the colored protein product with special optical properties (type and intensity of the color, and possibly color tones Image of colored turbidity), which are clearly dependent on a specific (determined) protein concentration in a liquid to be tested.

According to the invention, the test reagent contains the reagent mixture with the following weight%: dyes 0.01-0.03% by weight solvent 5.0-10.0% by weight citric acid 0.5-1.5% by weight urea 0.2-1.0% by weight sodium chloride 12-29% by weight hydrochloric acid 0.7-3.0% by weight water To 100% by weight

The dye used is preferably Coomassie Brilliant Blue G-250.

As a solvent, ethyl alcohol is preferably used.

The method for the detection of existing in liquids total protein is characterized in that the certain amount z. B. 500 microliter (μl) of a liquid to be tested (with transfer pipettes) in contact with the test reagent (1 ml) brings. Then after a short shaking (about three seconds) and a short exposure time, preferably after 40-60 seconds, you evaluate the result.

The result can be assessed visually, semi-quantitatively by comparison with a standard scale: digital monitor scale or pressed photo image scale ( - ).

The observation of colored samples and standards to be tested can be done in daylight as well as (more effectively) with electric lighting on a white background.

Investigations have shown that this protein test is suitable for semiquantitative determinations of protein present in liquids from 25 to 200 mg / L (eg for microproteinuria) and further up to 2,000 mg / L (without dilution). The result remains assessable for more than five hours.

With every photometer (with light wavelength 600 nm) or with optical computer program (eg PhotoM 121 http://softsearch.ru/programs/115-308-photom-download.shtml .
http://www.download.ru/soft/science/education/photom/6975 ) the result can be quantified.

This possibility - to be able to determine the protein content in such a broad concentration range (from 25 to 2,000 mg / L) without dilution of samples of the liquid first visually semi-quantitatively with a single analysis - is a great advantage. Here it is possible (specialists) optical density of the colored protein product from now or later with a photometer or with o. G. photometric computer program to achieve a quantitative analysis result.

As shown by lay studies, even unqualified people can flawlessly carry out the semi-quantitative procedure.

With this test you can quickly and easily thanks to clearly colored analysis products a protein determination -. As in urine tests - perform for Mikroproteinurien.

In the case of gum diseases, it has been possible with the protein test according to the invention (and in parallel with reference protein methods) to show that this tissue damage leads to an increase in the soluble protein content in the oral fluid (saliva).

Thus, the rapid test according to the invention (already the visual semi-quantitative method) preferably serves as the first warning signal, which shows as a pathological sign abnormally increased protein content in a liquid to be tested.

And in addition, you can visually read the result in a matter of seconds or hours.

Thus, the test reagent and the method are also suitable for their own use.

It is also possible to send the colored product images easily through various video systems to specialists for further analysis result evaluations, eg. As by e-mail, mobile phone, or Skype, etc., so that this test and the method can serve as a useful telediagnostic method already today.

Subsequently, the invention will be explained in more detail with reference to exemplary embodiments. Preferred embodiments are in the to shown:

Legend to the pictures

• 1) Lampe
• 2) Standardprobe

Schematic photo scale for protein determination in urine

• 1 ) Lamp
• 2 ) Standard sample

• 1) Lampe
• 2) Standardprobe
• 3) Urinprobe bei: Mikroproteinurie – links schwere Proteinurie – rechts

Photographic scale for protein determination and urine sample to be tested (below)

• 1 ) Lamp
• 2 ) Standard sample
• 3 ) Urine sample at: Microproteinuria - left heavy proteinuria - right

• 1) Lampe
• 2) Standardprobe
• 3) Speichelprobe von: Patient mit dem Gingivitis – links Patient mit dem Parodontitis – rechts

Schematic photo scale for protein determination in saliva and saliva sample to be tested

• 1 ) Lamp
• 2 ) Standard sample
• 3 ) Saliva sample from: Patient with gingivitis - left Patient with periodontitis - right

embodiments

example 1

Preparation of the photo scale and measuring scale in investigations with protein solutions.

The test reagent has the following composition: Coomassie Brilliant Blue G-250 0.02% by weight ethyl alcohol 5.0% by weight citric acid 0.5% by weight urea 0.2% by weight sodium chloride 27% by weight hydrochloric acid 1.5% by weight water 65.78% by weight

The test reagent - 1000 μl is contained in a reagent tube (length 55 mm, diameter 12 mm, made of polystyrene).

A series of solutions (containing 0.9% sodium chloride) of human albumin (A) at concentrations:
0 25 80 200 500 and 2000 mg / L prepared.

Method:

Add 500 μL of each protein solution to the reagent vial and shake briefly (about 3 seconds) the capped vial.

Then, after about 50 seconds, you observe and photograph (in daylight and under an electric table lamp) and save the colored samples as a monitor scale, you print the scale and get so two printed photo scales.

The following was stated under electric light, the colored protein products in the reagent vessel showed a special picture of the type and intensity of the color, as well as shades and possibly the image of colored turbidity, which are in clear dependence on the specific protein concentration in the liquid to be tested , This creates a new possibility for the objective visual determination of the protein content in liquids.

Then, these stained standard samples of o. G. Protein solutions were measured with photometer PF-11 (Machfrey-Nagel) at long wave 605 nm.

Results are on - and presented in Table 1. Table 1 Descriptions of images of the mixtures in the test tubes and measurement data after the reaction between the test reagent and the above-mentioned protein solutions Protein content mg / L .. by daylight Descriptions of pictures under table lamp in the dark Measurement data Optical density (average: 5 measurements) 0 (water) bright pink bright pink 0.210 25 bright purple purple 0,350 80 light gray-purple-green Purple violet 0,520 200 blue green violet, slightly clouded 0,850 500 blue blue-violet, clearly clouded 1,150 2000 blue bright turquoise, heavily clouded 1,490

The tests showed that the colored samples show more distinct visual differences in the case of electric light and the photo scale taken under the electric lamp than with the "daylight scale". As illumination, the table lamp may be a halogen or other lamp, or a polyfunctional pocket device (PTV) with LED light ( DE 20 2009 003 453 U1 ).

At the same time, the result (the picture) can be evaluated for more than five hours with this light.

It has also been found that with the test reagent according to the invention it is possible to determine visually (semi-quantitatively) in liquids to be tested the protein in the concentration range from 25 to 2000 mg / l without dilution.

However, in quantitative measurements under the same conditions linearity of optical curve only goes up to a protein concentration of 200 mg / L.

• 1) Man misst die Standardprobe mit ca. gleicher Eiweiß-Konzentration und gleiche Menge wie zu prüfende Probe (z. B. 500 μl von 2.000 mg/L);
• 2) Man muss weniger zu prüfender Flüssigkeit (z. B. 50–100 μl) eingeben und entsprechenden Standardproben verwenden.
• 3) Alternativ kann man im diesen Fall Probe verdünnen und dann mathematisch korrigieren.

So protein values from 200 mg / L (visual test shows) provide opportunities for proper protein content measurements:

• 1) Measure the standard sample with approximately the same protein concentration and the same amount as the sample to be tested (eg 500 μl of 2,000 mg / L);
• 2) Enter less fluid to be tested (eg 50-100 μl) and use appropriate standard samples.
• 3) Alternatively, in this case one can dilute the sample and then correct it mathematically.

Example 2

Examination of protein content in urine samples from diabetics

When examining protein in liquids to be tested, eg. As urine, add 500 .mu.l of these in a reagent vessel (as above) and then determine the protein content by comparing the reacted sample with the photo scale (if necessary with standard samples) or after photometric measurements.

The test reagents according to the invention were used to examine the urine samples of 15 patient diabetics from three medical practices specialized in the field of nephrology and diabetology, as described above.

Each sample was analyzed from 3 to 7 times in parallel.

Furthermore, this sample became parallel after DE-10 2004 046 326 A1 (as a reference method) checked. Several urine samples were tested and evaluated by five independent laypersons and, in addition, a statistical analysis of some measured results was performed.

Results are on and presented in Tables 2-4. Table 2 Determination of protein content in urine samples from diabetics No sample Picture Under the table lamp Protein content mg / L according to photo scale OD Measurements of protein content mg / L 1 Pink-purple clear under 25 0,320 23 2 Bright turquoise Heavily clouded about 2,000 1,440 1930 * 3 Purple clear about 25 0,400 31 4 Pink-purple clear under 25 not measured 5 Violet faintly clouded about 200 0.880 207 6 Pink-purple clear under 25 not measured 7 Pink-purple clear under 25 not measured 8th Violet faintly clouded about 200 0,810 190 9 Purple-violet clear about 80 0,500 77 10 Purple-violet about 80 0,650 100 11 Pink-purple clear under 25 not measured 12 Violet faintly clouded about 200 0,790 175 13 Purple-violet clear about 80 0,540 83 14 Bright turquoise heavily clouded about 2,000 1,590 2134 * 15 Pink-purple clear under 25 0,300 21 * The sample was calculated with the protein standard 2,000 mg / L. Table 3 The test results of some urine samples visually performed by five laymen with lamplight and image scale (protein content mg / L) Results of the provisions of the layman No result sample number 1 2 3 4 5 Measuring results 3 about 25 about 25 about 25 about 25 more than 25 31 12 about 200 about 200 about 200 about 200 between 80/200 175 14 about 2,000 about 2,000 about 2,000 about 2,000 about 2,000 2134 Table 4 Statistical analysis of accuracy of the protein assay of the present invention in parallel Examinations of a series of urine samples Raw data measurements (OD) No urine sample 1 2 3 4 5 6 X cv 3 0.38 0.37 0.41 0.42 0.40 0.42 0.40 5.24 12 0.75 0.78 0.87 0.79 0.80 0.76 0.79 5.4 14 * 1.60 1.70 1.55 1.63 1.62 1.50 1.60 4.31 Cv - Variation coefficient
* The sample was calculated with the protein standards 2,000 mg / L.

• 1) dass man mit erfindungsgemäßem Testreagenz visuell praktisch ohne Fehler die bedeutende Proteinurie ganz einfach bestimmen kann. Somit ist es zur Eigenanwendung geeignet.
• 2) Dass dieser Test bei Fotometrischen Messungen genügende Richtigkeit zeigt.

These tables show:

• 1) that one can determine visually virtually without error the significant proteinuria easily with inventive test reagent. Thus, it is suitable for self-application.
• 2) That this test shows sufficient accuracy in photometric measurements.

Almost the same accuracy was also observed in evaluations of the test results with optical computer program PhotoM 121. Almost the same protein levels in urine samples were shown using reference methods.

Example 3

Applications of the protein assay according to the invention to saliva tests in healthy persons and patients with periodontal disease.

The test reagent had a composition as in Example 1 and was in analogous test tubes

1) procedures and evaluations

First, a cotton swab was tested for suitability as a protein-liquid transmitter and it was shown that the wet rod transfers about 200 μl of saliva.

Then, a series of solutions (with the saliva of healthy people) and in addition with human albumin (A) was added with the addition of concentrations:
0, K (without addition A), 80, 200, 500 and 2,000 mg / L prepared in small beakers.

Then hold a cotton swab in the beaker (rotating) for about 20 seconds to thoroughly soak it with saliva and place it in the reagent vial with the test reagent and spin it there for about 30 seconds, then squeeze the stick on the vessel wall and discard it ,

Then, after about 50 seconds, the colored samples are photographed under the table lamp and saved as a monitor scale. You print them and get printed photo scales ( ).

Here, one measures the colored liquid in the vessel with the photometer (as in Example 1) to obtain a table for the quantitative determination of the protein content in saliva.

2) Investigations with the protein test according to the invention of the "vacuum saliva" of healthy people and patients with periodontal diseases (for example periodontitis, gingivitis and others).

Principle: For o. G. Disease increases the permeability of the tissue barrier strongly and soluble proteins, enzymes u. a. of tissues, inflammation products, as well as bacteria can spread rapidly in the oral cavity (in the saliva), thus reflecting the tissue damage condition to the periodontium.

Saliva samples from patients were obtained from 12 dentists (medical practices) specializing in periodontology.

• – Gesunde 17 junge Frauen 20–27 Jahre alt (Zahnarzthelferinnen) und 11 Männer 56–75 Jahre alt, die schon lange ohne Zähne, aber mit Prothesen leben;
• – Gruppe (15 Männer – einschließlich Zahnärzte und 3 Frauen) die verschiedene Stadien von Gingivitis haben;
• – Gruppe (9 Männer 42–53 Jahre alt) mit Parodontitis.

Several groups were tested:

• - Healthy 17 young women 20-27 years old (dental assistants) and 11 men 56-75 years old, who have long lived without teeth, but with dentures;
• - Group (15 men - including dentists and 3 women) who have various stages of gingivitis;
• - Group (9 males 42-53 years old) with periodontitis.

Instructions:

Requirement:

No invasive oral treatment within the last 2 days before the test and no brushing within the last 6 hours!

The patient rinses his mouth twice with water and spits it out. Then, for the saliva secretion, he touches with his tongue a lemon slice and holding for about 30 seconds the mouth sucking (with vacuum), and during this time he makes about 7-9 times movements for "inner flushing" of the closed oral cavity and then there he (spits) the saliva in the small cup.

Then the patient holds a cotton swab for about 20 seconds in the cup or directly in the mouth (rotating) to thoroughly soak it with saliva and put it in a reagent container with the reagent mixture (pink colored) and turns it there for about 30 seconds, Thereafter, the rod is squeezed out of the vessel wall and discarded.

Then shake briefly the closed vessel and from about 50 seconds to measure the colored liquid in the vessel with the photometer (at 600 nm) and determines the quantitative protein content in saliva.

Alternatively, one can compare the colored liquid in the vessel with the monitor scale or the printed photo scale and (semi-quantitatively) determine the protein content.

The result - the colored liquid - remains assessable for a few hours

In parallel, the determination of the protein content with precipitation reaction was carried out in saliva samples to be tested

Results are in and Table 5 presented: Table 5 Total protein content in saliva samples from patients with various paradont diseases in guantitative studies with protein assay according to the invention group Concentration mg / ml healthy 31 (0.09 to 0.3) 0.15 gingivitis 18 (0.47 to 0.85) 0.58 periodontitis 9 (1.30 to 3.00) 1.82 n X range (X-average).

The table and deviations show that the protein test according to the invention allows for various gum states to clearly and instrumentally determine the total protein content in the saliva. At the same time at successful treatment it is observed in some cases decrease in the raised protein values.

The precipitation reaction method showed almost the same results.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• RU 2077060 C1 [0003]
• DE 10030193 A1 [0003]
• DE 10144436 A1 [0003]
• DE 202009003453 U1 [0037]
• DE 2004046326 A1 [0045]

Cited non-patent literature

• Bredford, Anal Biochem. 72, 1976 [0004]
• http://softsearch.ru/programs/115-308-photom-download.shtml [0016]
• http://www.download.ru/soft/science/education/photom/6975 [0016]

Claims (11)

Test reagent for a rapid test for protein determination in biological fluids, in particular in urine, comprising a reagent mixture and a reagent vessel, characterized in that the reagent mixture additionally contains urea, citric acid, hydrochloric acid and sodium chloride in addition to a conventional dye and a solvent. Test reagent according to claim 1, characterized in that it contains the reagent mixture with the following% by weight: dyes 0.01-0.03% by weight solvent 5.0-10.0% by weight citric acid 0.5-1.5% by weight urea 0.2-1.0% by weight sodium chloride 12-29% by weight hydrochloric acid 0.7-3.0% by weight water to 100% by weight Test reagent according to claim 1 or 2, characterized in that as a dye Coomassie Brilliant Blue G-250 is included. Test reagent according to one of claims 1 to 3, characterized in that the solvent is ethyl alcohol. Test reagent according to one of claims 1 to 4, characterized in that the reagent mixture is prepared so that it serves as a visually analytical indicator, wherein the interaction of color reaction and turbidity reaction, a colored protein product is formed with special optical properties, which in clear dependence of the protein concentration in the liquid to be tested. Test reagent according to one of claims 1 to 5, characterized in that it is coupled to an illumination source, whereby the formed and colored protein product is displayed. Test reagent according to claim 6, characterized in that the illumination source is a table lamp with halogen or another lamp or a polyfunctional pocket device (PTV) with LED light, thereby visually in the liquid to be tested, the protein in the concentration range of 25 to 2000 mg / L. can be determined without dilution. Test reagent according to one of claims 1 to 7 usable in a method for the detection of total protein present in liquids, wherein a certain amount of liquid to be tested is brought into contact with the test reagent and then after a short reaction time, preferably after 40-60 seconds, the result by comparison is rated with the standard scale. Test reagent according to Claim 8, characterized in that the result of the total protein determination is produced in the form of a digital monitor scale or printed photo image scale which is evaluated (Example 1, - ), whereby the result (colored sample) is maintained for at least five hours. Test reagent according to claim 8, characterized in that the result of the total protein determination is quantified with a photometer (with light wavelength 600 nm) or with an optical computer program (eg PhotoM 121, Example 1). Test reagent according to one of Claims 1 to 10, characterized in that it has a liquid transfer agent (for example a cotton swab) for transferring a liquid to be tested (for example saliva) into the reagent vessel.

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