DD216806A1 - TEST STRIPS FOR THE DETECTION OF PROTEINS IN URIN - Google Patents

Abstract

The field of application of the invention is the laboratory diagnostics in the field of health care. The aim is a test strip for the detection of proteins in urine. The test strip consists of a carrier material which has been impregnated with a color indicator and buffering agents. Optionally, other ingredients are agents for preventing washout effects, such as water-soluble cellulose derivatives, aftertreatment agents such as acridine orange, true yellow or dipyridamole, and surfactants such as sodium dodecyl sulfate, chaotropic substances such as potassium thiocyanate or lithium chloride, and denaturants such as urea or guanidine hydrochloride as modifiers of protein susceptibility sensitivity. The test strip is characterized by a high sensitivity. Low protein concentrations, d. but already about d. usually m. d. Urine excreted protein amount are m. a clearly visible color displayed. With the urine of healthy persons no false-positive results are obtained.

Description

• Test strips for the detection of proteins in the urine

Field of application of the invention

The invention relates to test strips for the detection of proteins in urine. The test strips are indicated in the laboratory diagnostics t. , ·

Characteristic of the known technical solutions

The study of urine on proteins belongs to every medical. Basic examination. for early detection of kidney disease h nits. The detection of proteins is also useful for the follow-up of patients with kidney disease and for. the timely detection of! animal damage following the administration of some medicines, such as As antibiotics and cytotoxic agents of importance. '

Older methods for the detection of proteins in urine are today only occasionally carried out cooking with acetic acid and the HELLSR ring sample with nitric acid. Widely used in clinical laboratories is the sulfosalicylic acid test. 'The method is sufficiently sensitive and detects all plasma proteins that can occur in the urine with the same sensitivity. Oral antidiabetics, high doses of antibiotics, p-aminosalicylic acid and X-ray contrast agents interfere with the response. .An disadvantage certain amount of effort (centrifuging or filtering the urine ¹ and preparation of the reagents, cleaning of glassware) (P. Balint (eds). Clinical 'laboratory diagnostics, 3d 1. People and Health, Berlin 1962, page 284th) ..

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For the detection of a proteinuria also test strips are used. They are impregnated with a pH indicator which reacts with a color change above a defined protein concentration. "The reaction zone is yellowish or pale, greenish in its dry state and should display proteins with green to blue 'color' depending on their concentration However, after immersing in a test solution, a greenish tint often occurs, even if the sample is protein-free, making it difficult to distinguish negative from weak positive reactions.

Object of the invention . · -.-.

The aim of the invention is to improve the uniqueness of the color reaction in the detection of proteins in urine by means of test strips.

Explanation of the essence of the invention

The object of the invention is to develop a test strip with an absorbent carrier for the color indicator. '·. '

According to the invention, the deposit is achieved by a test strip for detecting proteins in urine on the basis of absorbent materials such as paper or nonwovens. The test strip according to the invention is characterized in that it contains as color indicator a compound of the general formula I (see Appendix), and Buffering agent, wherein as a buffer citric acid / phosphate, citric acid / UaOH, or succinic acid / I ¹ TaOH were used. Furthermore, the strip is optionally admixed with agents for preventing washout effects such as water-soluble cellulose derivatives, optionally colorants for background dyeing and optionally surfactants, ohaotropic substances or denaturants for modifying the sensitivity.

In case of a proteinuria due to a leuphropathy, mainly albumins are eliminated. 7 / eiterhin _v are essential in ¹ lent lower concentrations the antibody IgM, IgG and IgA, lambda and kappa chain, transferrin, haptoglobin, - lysozyme before and several globulin fractions. For the diagnosis and differential diagnosis of leu- phathathias it is sufficient if albumin, as the main constituent of the proteins in the urine, detects

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is rejected and the globulins and other Proteinfrak- ^': functions are listed only to a lesser extent, as the excretion of albumin is proportional in most cases to total protein.

The proteins, but mainly the albumin, bind color indicators of the general formula I (see Appendix) in their anionic form. In this binding, the color indicators show a color change towards the Salaform, although the pH of the environment remains constant (indicator error). Such color indicators are chosen which show a clear color change. The salts occurring in the urine should not influence the color.

The -pH value of the buffer used for the impregnation depends on the transition point of the dye indicator of the general formula I '(cf. Appendix). Preferably, such dyes are incorporated in the vehicle whose two transition points are in the acidic pH range. Inventive examples of the color indicators are m-cresol purple, p-cylenol blue, thymol blue, bromochlorophenol blue, bromophenol blue, bromcresol green, ghlorophenol red, bromophenol red, bromocresol purple and bromothymol blue. The pH of the buffer should be close to the acidity of the acidic transition point of the dye indicator.

The acidic pH range is also chosen because the color indicators show maximal binding to the proteins in the acidic environment, and it is further advantageous that bilirubin, possibly bound to albumin and excreted in the urine, is liberated at acidic pH values. so that color indicator and bilirubin do not compete for the binding sites on the protein molecule, _λ

The buffer must have a strong buffering capacity. Buffering prevents the 0H ~ and H ions in the urine from altering the pH of the impregnated carrier, after moisturizing, and possibly causing an inverted staining indicator even though no protein is present in the urine. , ,

The washing out of the buffering agent and the dyeing indicator is prevented by adding the impregnating solution to

if water-soluble cellulose derivatives such as e.g. Cellulose sulfate or carboxymethyl cellulose are added. As a result, the buffer capacity of the impregnated carrier remains constant after immersion in the urine and it is avoided that the buffer components washed out and the color indicator interfere with further examinations which are to be carried out with the same urine sample after the proof. ·

The concentration of the dye indicator of the general formula I (see Appendix) depends on the pH of the buffering agent and the carrier material used. In a carrier with a relatively thick layer thickness, 'z. 3. Chromatography paper, and at a pH close to the acidic turnover point, the impregnation may be carried out with a lower. Color indicator concentration take place. In this case, protein-free urine or urine from healthy persons with a very low protein concentration results in a clearly negative reaction. A possible color change is then recognized and is safe. due to a higher (pathological) Protein concentration ¹ · If one chooses the other hand, a higher color indicator concentration and a smaller layer thickness of the carrier ·, so ', already occurs auch'bei a low protein concentration, a slight coloration on ,. which increases as the amount of protein increases. However, different intensities of a hue are more difficult to differentiate than a hue from one hue to another. ^! , '

Therefore, the composition of the impregnating solution of the receiving volume of the carrier material and of the. Indicator error of the dye indicator dependent. The most favorable variant is a support with a high absorption volume, which has been treated with a _% impregnation solution of low color indicator concentration. '

Optionally, a background dyeing of the paper with yellow dyes can be carried out together with the impregnation or subsequently. True yellow, acridine orange and dipyridamole have proven successful. The background coloration further improves the distinctness between negative and weakly positive reactions.

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Sensitivity of the test strip can be increased by adding to the impregnating solution compounds that reduce or "dissolve" hydrogen bonds, including surfactants such as ITatriumdodecylsulfate, chaotropic substances such as potassium rhodanide and lithium chloride, and the denaturants urea and guanidine Depending on the concentration, these compounds develop or denature the proteins and the number of binding sites for the color indicator molecules increases, resulting in a more intense coloring even at a low protein concentration in the urine.

The test strip according to the invention is distinguished by a distinctly different coloration as a function of the provitein concentration in the urine. The addition of denaturants as well as surfactants and chaotropic substances increases the sensitivity of the test strips by about 50 % . No false-positive urine results were obtained from healthy subjects. · ''

embodiment

20 mg of bromophenol blue are dissolved in 100 ml of Mcllvain's citric acid / phosphate buffer with a pH of 2.7. Tailored chromatography paper is placed in this solution and, after removal and draining, dried at room temperature Paper is dyed yellow ..

The impregnated. Paper is in longitudinal strips with a ·. Width of 1 cm cut and glued on a plastic film. This is cut into strips of 0.5 cm width. Each test strip has a reaction zone of 0.5 x 1 cm.

To test the urine for proteins, the test strip is immersed in the test solution for a few seconds and the uninjected urine is removed by stripping off the lateral edge at the edge of the vessel. The reaction will be after. Read 30 to 60 seconds. A slight blue coloration occurs in the presence of about 30 mg serum protein / 100 ml urine. Higher concentrations cause a more intense blue coloration. ,

2. Dissolve 100 ng Bromcresolgreen sodium salt in 100 ml of a 0.25 molar citric acid solution adjusted to pH 3.5 with ITaOH. The further procedure in the impregnation and production of the test strip. \ fen takes place as described in Example 1. The test strip also has similar properties,

3. Dissolve 80 mg of bromcresol purple in 100 ml of a 0,10 molar succinic acid solution adjusted to pH 4,7 with HaOH. .The paper dried after impregnation is dyed yellow and, after being immersed in protein-containing urine, turns purple depending on the concentration of the proteine.

4. The strip is prepared as described in Example 1, except that 500 mg of cellulose sulfate are added to the impregnating solution. The strip thus obtained shows a slight color enhancement when immersed in proteinaceous urine, since the color indicator and the buffer do not diffuse into the test sample.

5v The strip is prepared as described in Example 1, but the impregnating solution is additionally _: 50 mg., Acridine orange added. The strip obtained in this way is strongly yellow in color, so that a clear distinction from the dyeing is obtained a protein concentration of about 25 mg / 100 ml of urine occurs is possible.

,. 6 The strip is prepared as described in Example 1, but 2 g of xiumium rhodanide are also added to the impregnating solution. The strip obtained in this way is yellowish in color and indicates a protein concentration of about 15 mg / 100 ml of urine,

7. D: The strip is prepared as described in Example 1, but 30 g ^ G-uanidinhydrochlorid are also added to the impregnating solution. The strip obtained in this way is slightly yellowish in color and indicates a rrotein concentration of about 15 mg / 100 ml of blue-colored urine. ·,. \ '

Plant ,

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R ₁ = R _Q = H, alkyl

R ₂ = R ₅ = Rg = R ₃ = H, Cl, Br ,, alkyl, isopropyl

H, 'OH. ·;.

-R ₇ = H, -.01, .Bi ,. Alkyl. ··· = Ξ, 'GOOH, _r SO ₃ H = H, OH ·.

R ₃ = R ₄ =

Claims (2)

Claim of invention , 1. Test strips for the detection of proteins in urine on the basis of absorbent materials such as paper and nonwovens. characterized in that the strip as an indicator of a compound of the general formula I (see Appendix), buffering agents, optionally agents for preventing Auswascheffekten such as water-soluble cellulose derivatives, optionally means for background dyeing as Schtlichtgelb, acridine orange or dipyridamole and optionally surfactants such as ITatriumdodecylsulfat , chaotropic substances 7, ie potassium thiocyanate or lithium chloride and denaturants,. such as guanidine or urea to modify the sensitivity. , 2. Test strip according to item 1, characterized in that the buffering agent is in particular citric acid / phosphate, citric acid / IJaOH or succinic acid / 3aOH. , '