JPH0564745B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to a test piece for measuring protein, particularly urinary protein. [Background of the Invention] It is said that even healthy people excrete 20 to 80 mg of protein in the urine every day, and the excreted protein is usually mainly albumin, which has small particles and easily passes through the glomerulus. On the other hand, if hemolysis is severe and a large amount of hemoglobin from red blood cells leaks into the plasma and this leaks from the renal glomerulus, or if there is inflammation in the kidneys or urinary tract, white blood cells are released into the urine, so globulin is the main component. This results in urine protein. Urine protein generally becomes high in the following cases and is an important indicator of kidney disease. (1) Acute and chronic nephritis, nephrosis. (2) Renal congestion due to heart failure, etc. (3) Febrile proteinuria. (4) Chemical poisoning, bacterial poisoning. (5) Leukemia, purpura. (6) Stricture, stone Obstruction of the ureter due to tumor. (7) Brain tumors, epilepsy, other central nervous system diseases, and mental disorders. (8) Contamination with pus, blood, semen, etc. (9) Intake of large amounts of low molecular weight proteins such as eggs. (10) A temporary condition that appears after strenuous exercise or prolonged exposure to hot or cold water. (11) Postural and juvenile proteinuria. Currently, the following methods are commonly used for measuring urine protein. (1) Sulfosalicylic acid method (sensitivity 0.002%) Clear urine 4-5ml + sulfosalicylic acid 20W/V%
2-3 drops of solution → If white turbidity or precipitation occurs, it is protein positive. (2) Boiling test method (sensitivity approx. 0.005%) Boil 5 ml of clear urine for 1 to 2 minutes. If turbidity occurs, add 1 to 3 drops of 5% acetic acid or 70% nitric acid when hot, and remove the turbidity. If unchanged or increased, protein is positive. (3) Robers method (sensitivity 0.003%) If the sample and a nitric acid solution of magnesium sulfate are mixed in equal volumes and a white ring forms at the interface, it is positive for protein. (4) Test paper method (sensitivity 0.03%) This method uses a protein error method in which pH indicators such as bromophenol blue or tetrabromophenol blue develop an alkaline color due to the presence of protein. (5) Coomassie brilliant blue method (sensitivity 0.001%) A highly sensitive measurement method that uses the binding of dye and protein.
50 μ + 3 ml of CBB-G250 solution → Measure the absorbance at 595 nm. (6) Biuret method using trichloroacetic acid precipitation Mix 2 ml of sample (original urine) + 2 ml of distilled water + trichloroacetic acid (10% solution), centrifuge at 3000 rpm for at least 5 minutes, and then discard the supernatant. Biuret's reagent (NaOH4
%, potassium sodium tartrate crystals 4.5%,
CuSO _{4.5H 2} O 0.5%, potassium iodide 0.5%)
After mixing 2 ml + 2 ml of distilled water, heat at 37℃ for 30 minutes.
Colorimetric at 540nm. Among these, the sulfosalicylic acid method, the boiling test method, and the Robers method require a large amount of sample by nephelometric method or a method similar thereto, and there are limits to their accuracy when applied to quantitative analysis. On the other hand, the Coomassie Brilliant Blue method is a colorimetric method, but it is considered unsuitable for continuous processing of a large number of samples because of the curvature of the calibration curve and the contamination of cells, test tubes, etc. Furthermore, the Biuret method using trichloroacetic acid precipitation requires a precipitation separation operation, which is complicated and impractical. In addition, although the test strip method using the protein error method is very simple and widely used, it has low sensitivity and is particularly difficult to judge between normal and abnormal. The drawback was that the coloration was extremely low. On the other hand, according to Analytical Chemistry Vol. 32 379-386 (1983), when protein (albumin) is added to a mixture of pyrogallol red and molybdate (PR-molybdenum complex), λmax shifts to the longer wavelength side and the protein is increased. There is a description that it is possible to perform colorimetric determination with sensitivity. However, when protein concentration is measured using the reagent described in the literature as a sample in the urine of a healthy person (negative by the sulfosalicylic acid method), the absorbance of most samples is lower than that of the reagent blank, and a negative value is calculated as the protein concentration. Encounter a phenomenon. Therefore, it is completely impossible to apply this method as it is to the measurement of urine protein using human urine samples as a sample, and there is a current need for a simple, highly sensitive, and highly accurate method for measuring urine protein. be. [Object of the Invention] In view of the above-mentioned current situation, the present invention aims to provide a simple, highly sensitive, and highly accurate method for measuring urine protein.
This was developed for the purpose of practical application of a urine protein measurement method using a urine dipstick method using molybdate and a dye that forms a complex with molybdenum and whose wavelength shifts in the presence of protein. [Configuration of the Invention] The present invention provides a molybdate, a dye that forms a complex with molybdenum, and whose wavelength shifts due to the presence of a protein, and a chelating agent that binds to molybdenum, or a dye that does not react with the dye, and which A test for protein measurement consisting of an absorbent carrier impregnated with a composition for protein measurement consisting of a buffer and a metal ion capable of binding to oxalic acid, citric acid, phosphoric acid, and their salts, which are expected to coexist in the carrier. This is a piece of invention. That is, as a result of extensive research into the practical application of a method for measuring urine protein that uses a dye that forms a complex with molybdenum and whose wavelength shifts due to the presence of protein, the present inventors have found that a method for measuring urine protein that uses a dye that forms a complex with molybdenum and whose wavelength shifts due to the presence of protein has been developed. Either a chelating agent capable of binding molybdenum is added to the measurement reagent in advance, or it does not react with the dye,
Oxalic acid, citric acid, which are expected to coexist in the sample,
The purpose can be achieved by adding phosphoric acid and metal ions that can bind with these salts, and it can be easily achieved by using a test piece consisting of a carrier (reagent holding piece) impregnated with such a reagent composition. The inventors have now completed the present invention by discovering that it is possible to measure urine protein with high sensitivity and precision. The present invention utilizes the fact that pigments such as pyrogallol red and pyrocatechol violet form complexes with molybdenum, and that this complex binds to proteins and shifts the maximum absorption wavelength to the long wavelength side. When measuring protein using the test strip method, chelating agents that are present in the sample and cause the color to fade, such as organic acids and/or phosphates, or chelating agents that have the same effect as these, must be used. The impregnating reagent contains metal ions that have been added in advance to the impregnating reagent, or can bind to oxalic acid, citric acid, phosphoric acid, and salts thereof that do not react with the dye and are expected to coexist in the sample. By adding globulin to albumin, it is possible to avoid the phenomenon in which the color of normal urine fades, and to measure urinary protein with extremely high accuracy. This makes it possible to measure. In other words, pigments such as pyrogallol red and pyrocatechol violet form complexes with molybdenum and become colored (or their coloring increases), but when other chelating agents that have binding strength with molybdenum coexist, some of the molybdenum is The moiety separates from its binding with these pigments and binds with other chelating agents, resulting in a dimming of the blind coloration. As the chelating agent is gradually added to the impregnating reagent, the color of the blind test will gradually become lighter depending on the amount added, and beyond a certain amount, even if the chelating agent derived from the sample is mixed in, it will no longer be that color. The phenomenon in which the coloration becomes lighter is hardly observed, and the coloration becomes less pale. Similarly, when aluminum ions, cerium ions, etc. are added to the impregnating reagent, the chelating agents (oxalic acid, citric acid, phosphoric acid, etc.) in the sample bind aluminum ions, cerium ions, etc. and do not bind to molybdenum ( (or the binding ratio decreases), and it is possible to avoid the color from becoming pale. Therefore, by adding in advance a chelating agent capable of binding to molybdenum or metal ions such as aluminum ions and cerium ions to the measurement composition, the phenomenon in which the color of normal urine becomes too pale can be resolved. At the same time, accurate measurement is possible even when measuring a sample containing a high concentration of protein using a reagent containing a chelating agent that binds to molybdenum, aluminum ions, cerium ions, etc. The present inventors discovered a previously unexplained cause of negative values in normal urine in a urine protein measurement method that uses a dye that forms a complex with molybdenum and whose wavelength shifts in the presence of protein. We investigated the
As a solution to this problem, the present inventors drew the above conclusion based on their own knowledge and arrived at the present invention. According to the method of the present invention, the coloration of normal urine does not become too pale, and both quantitative performance and reproducibility are good. Furthermore, since the test piece for protein measurement of the present invention is highly sensitive, it can be used to detect the borderline between normal and abnormal (protein concentration 10 to 30 mg / dl) became clearer and easier to judge. In addition, in the conventional protein error method, the coloration of globulin relative to albumin was extremely low;
In the method of the present invention, globulin is colored to almost the same extent as albumin, making it possible to detect globulinuria, which may have been overlooked by conventional test strip methods. As the chelating agent used in the present invention,
Ethylenediaminetetraacetic acid (EDTA), hydroxyethylethylenediaminetriacetic acid (EDTA-OH),
Ethylenediaminediacetic acid (EDDA), iminodiacetic acid (IDA), nitrilopropionic acid (NTP), nitrilotriacetic acid (NTA), hydroxyethyliminodiacetic acid (HIDA), citric acid, tartaric acid, oxalic acid, 1-hydroxyethane-1 , 1-diphosphonic acid, pyrophosphoric acid, hexametaphosphoric acid, tripolyphosphoric acid, metaphosphoric acid and the like. These may be used alone or in a mixture of two or more, and in order to improve the dissolution process when preparing the impregnating solution, they may be added in the form of salts such as alkali metal salts such as sodium, potassium, and lithium, or ammonium salts. It's okay. The amount added varies depending on the chelating agent added, but it is usually impregnated into an absorbent carrier such as paper or nonwoven fabric in one to several portions at a concentration of 0.001 to 1%. Also, suitable metal ions that can be used in the present invention include aluminum ions and cerium ions. Dyes that can be used in the present invention include pyrogallol red (PR), brompyrogallol red, pyrocatechol violet (PV), and o-hydroxyhydroxy nymph, which quantitatively form a complex with albumin in the presence of molybdenum. Examples include pigments such as talen and gallein. The amount of these pigments used is usually 0.0005 to 0.1 as the concentration of the impregnating solution.
%, and may be impregnated into an absorbent carrier such as paper or nonwoven fabric in one to several portions. Molybdenum that forms a complex with these pigments is usually used as an aluminum salt or an alkali metal salt of molybdic acid, but is not limited to these. For example, in the case of molybdate, the concentration of molybdate ion in the impregnating solution is usually about 0.0005 to 0.1%.
The absorbent carrier can be impregnated in one to several batches. Absorbent carriers used in the test strip for protein measurement of the present invention include, without exception, absorbent carriers used in ordinary test strips for measurement, such as paper, cellulose, chemical fibers, synthetic resin woven fabrics, and non-woven fabrics. used. In addition, as a support for holding an absorbent carrier,
Examples include glass fiber, and synthetic polymer compounds such as polyvinyl chloride, polystyrene, polyvinyl acetal, acetylcellulose, nitrocellulose, polyvinylidene chloride, and polypropylene, and sheets of these or coated cardboard are preferably used. There are no particular restrictions on the size, dimensions, etc. of these absorbent carriers and supports, and as long as they are used in accordance with the size and dimensions of the absorbent carriers and supports in commonly used measurement test pieces. Enough. Regarding the method of impregnating the drug into the absorbent carrier and the method of adhering the absorbent carrier to the support, any method used for ordinary measurement test pieces may be used, or special methods may be used. No method required. Table 1 shows the effect of adding a chelating agent in the method of the present invention. The measurement conditions are as follows. Measurement conditions Sample urine Protein negative (by sulfosalicylic acid method) Urine A,
B and C contain human albumin or human γ-globulin.
The sample was added at a concentration of 20 mg/dl. Impregnation liquid: Concentration of pyrogallolred 250mg/, sodium molybdate 300mg/
It was dissolved in 0.5M glycine-hydrochloric acid buffer (PH2.5). Method for Preparing Test Pieces Various chelating agents were added to the above impregnating solution at the concentrations shown in the table, and paper with a thickness of 0.4 mm was impregnated with this and dried. The test paper thus obtained was cut into 5 mm squares and attached to one end of a 5 mm x 8 cm polyvinyl chloride sheet using double-sided adhesive tape to form a test piece. Measurement method This test piece was momentarily immersed in sample urine, and the color development after 60 seconds of impregnation was measured. The coloration was investigated by comparing the coloration of negative urine and the coloration of urine to which human albumin or γ-globulin had been added.

【table】

[Table] As is clear from Table 1, the addition of the chelating agent causes the test strip to change color due to the protein in the urine, making it possible to measure protein. Further, Table 2 shows the effect of adding aluminum ions and cerium ions in the method of the present invention.
The measurement conditions are as follows. Measurement conditions Sample urine Same as in Table 1. Impregnation liquid Same as in Table 1. Preparation method of test piece Add cerium acetate (0.05%) or aluminum sulfate (0.05%) to the above impregnating solution, and add this to the thickness
It was impregnated onto 0.4 mm paper and dried. The test paper thus obtained was cut into 5 mm squares and attached to one end of a 5 mm x 8 cm polyvinyl chloride sheet using double-sided adhesive tape to form a test piece. Measurement method: Same as in Table 1.

〔Example〕

Example 1 (1) Preparation of impregnating solution Pyrogallol red 25mg Ammonium molybdate 30mg Oxalic acid 300mg Glycine 3.75g Sodium chloride 3g Dissolve these in 90ml of water, adjust the pH to 2.5 with hydrochloric acid,
The total volume was 100ml. (2) Preparation of test piece Kutomato paper (thickness 0.4 mm) was impregnated with the above impregnation liquid and dried. The thus prepared test piece was cut into a 5 mm square, which was attached to a polyvinyl chloride sheet (5 mm x 8 cm) using double-sided adhesive tape to form a test piece. (3) Measurement method and measurement results After the test piece was momentarily immersed in the sample urine and impregnated, color development was observed about 1 minute later. The test paper portion was colored from bluish-purple to blue depending on the protein concentration. The detection limit is
It was 10mg/dl. Example 2 (1) Preparation of impregnating solution Pyrogallol red 25mg Ammonium molybdate 30mg Citric acid 600mg Glycine 3.75g Sodium chloride 2g Dissolve these in 90ml of water, adjust the pH to 2.5 with hydrochloric acid,
The total volume was 100ml. (2) Preparation of test piece A test piece was prepared in the same manner as in Example 1. (3) Measurement method and measurement results Measurement was carried out in the same manner as in Example 1, and the same results were obtained. Example 3 (1) Preparation of impregnating solution Pyrogallol Red 25mg Ammonium Molybdate 30mg EDTA・2Na 100mg Glycine 3.75g Dissolve these in 90ml of water and adjust the pH to 2.5 with hydrochloric acid.
The total volume was 100ml. (2) Preparation of test piece A test piece was prepared in the same manner as in Example 1. (3) Measurement method and measurement results Measurement was carried out in the same manner as in Example 1, and the same results as in Example 1 were obtained. Example 4 (1) Preparation of impregnation solution Pyrocatechol violet 10mg Ammonium molybdate 30mg EDTA・2Na 100mg Glycine 3.75g Potassium chloride 2g Dissolve these in 90ml of water, adjust the pH to 2.5 with hydrochloric acid,
The total volume was 100ml. (2) Preparation of test piece A test piece was prepared in the same manner as in Example 1. (3) Measurement method and measurement results After the test piece was momentarily immersed in the sample urine to impregnate it, color development was observed about 1.5 minutes later. The test paper area turned green depending on the protein concentration. The detection limit is 10
It was mg/dl. [Effects of the Invention] As described above, the present invention provides an improved method for measuring protein, especially urine protein, using a test strip method. Unlike the test strip method based on the protein error method described above, this invention has a remarkable effect in that it is a measuring method that can also detect γ-globulin, and it is an extremely significant invention that contributes to this industry.

Claims (1)

[Claims] 1. A molybdate, a dye that forms a complex with molybdenum and whose wavelength shifts due to the presence of protein, and a chelating agent that binds to molybdenum, or a chelating agent that does not react with the dye and is present in the sample. A test piece for measuring protein comprising an absorbent carrier containing a composition for measuring protein comprising a buffer and a metal ion capable of binding with oxalic acid, citric acid, phosphoric acid and salts thereof, which are expected to coexist. 2. The test piece according to claim 1, wherein the protein is urinary protein. 3 The chelating agent is ethylenediaminetetraacetic acid (EDTA), hydroxyethylethylenediaminetriacetic acid (EDTA-OH), ethylenediaminediacetic acid (EDDA), iminodiacetic acid (IDA), nitrilopropionic acid (NTP), nitrilotriacetic acid (NTA),
Hydroxyethyliminodiacetic acid (HIDA), citric acid, tartaric acid, oxalic acid, 1-hydroxyethane-1,1-diphosphonic acid, pyrophosphoric acid, hexametaphosphoric acid, tripolyphosphoric acid, metaphosphoric acid, and one of these salts or 2 or more types of test pieces according to claim 1 or 2. 4. The test piece according to claim 1 or 2, wherein the metal ions are aluminum ions and/or cerium ions. 5. The test piece according to any one of claims 1 to 4, wherein the dye is pyrogallol red or pyrocatechol violet.