JPH0798318A - Measuring method and measuring tool for protein in urine - Google Patents

Abstract

PURPOSE:To readily detect the protein other than albumin by impregnating an absorbing carrier with a buffer giving the acidic condition, a dye generating a color tone change under the existence of the protein, and & material generating heat with an aqueous sample, and reacting it with the aqueous sample at a specific reaction temperature. CONSTITUTION:The reaction temperature is preferably set to the range of 30-50 deg.C, and the reactivity of the protein other than albumin, particularly Bence- Jones protein, is remarkably improved in this temperature zone. The reaction condition of 30-50 deg.C is obtained by the following methods: a material generating heat when reacted with urine is mixed, a heated sample is used, the sample is reacted in the preset temperature environment, or the test is conducted on a heated plate. Calcium chloride, calcium bromide, magnesium chloride, zinc bromide, or zinc chloride is used for the material generating heat when reacted with urine, for example.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel method for measuring protein in urine, and more particularly to a method and a device for measuring protein in urine using a pigment which causes a color change due to the presence of protein.

[0002]

2. Description of the Related Art Urine protein can be measured by measuring the turbidity produced by adding sulfosalicylic acid or trichloroacetic acid to urine by a nephelometric method, or by using a test strip utilizing the protein error of pH indicator, There are methods for measuring protein fractions by electrophoresis, immunological methods, etc., but it is common to screen using a method with a test strip that is excellent in simplicity, and to accurately examine only abnormal samples by electrophoresis etc. Target.

By the way, even if the measuring method used for the test paper says "Measurement of protein in urine", it actually reacts with albumin but hardly reacts with proteins other than albumin. . The protein error method used in the urine protein measurement test is that when albumin is added to a pH indicator under acidic conditions, the pH indicator and albumin bind to each other and the color tone of the pH indicator changes to be alkaline. Is used. Proteins other than albumin do not have the same sensitivity as albumin, because they have a weak effect on changing the color tone of the pH indicator.

Proteins excreted in urine include albumin, various globulin, Bence Jones protein, mucoprotein, and the like. For example, in the case of an albuminemia, proteins other than albunmin are excreted with urine. Further, in the case of causing neoplastic growth of immunoglobulin-producing cells such as multiple myeloma and primary macroglobulinemia, the Bence Jones protein is particularly contained in a large amount. However, these abnormalities could not be found by conventional screening using a urine test strip that mainly detects albumin.

[0005]

In view of the above drawbacks, the present inventors have proposed a method for measuring a urinary protein having high reactivity with a protein other than albumin, which has a simple protein error method for detecting a protein in urine, and It is an object to provide a test strip for measuring protein in urine.

[0006]

Surprisingly, it has been found that the above problems can be achieved extremely easily. Measure temperature at 3
This is because it was discovered that the reactivity of proteins other than albumin, especially Bence Jones protein, was particularly enhanced at 0 to 50 ° C. Further, in order to achieve the reaction condition of 30 to 50 ° C., the above-mentioned problem can be solved by mixing a substance that generates heat with urine, for example, a substance that dissolves in urine and generates heat in the reaction part of the test paper.

[0007]

Since albumin in urine reacts very quickly with a pH indicator, no attempt has been made to improve the reactivity by heating. Therefore, it was surprising that the protein error caused by the proteins other than albumin is increased by heating. Especially, it was surprising that the reactivity of Bence Jones protein was particularly improved by heating. Although the cause is unknown, unlike other proteins, Bence Jones protein may begin to precipitate at around 56 ° C when heated, and thus the quaternary structure may change significantly depending on the temperature.
As a result, the protein error is estimated to increase.

The pH indicator used in the present invention is disclosed in JP-B-45.
-1873, JP-A-60-97274, JP-B-42-1
Any of those which produce an alkaline error by reacting with a pH indicator described in 638 etc., tetrabromophenolphthalein ester, methyl orange, bromcresol purple, tetrabromophenol blue, bromcresol green, bromthymol blue Etc. are mentioned as a typical example.

As a buffering agent which gives an acidic condition, 0.1 to 1.
It gives the lowest pH of 0, and the buffering action of urine causes the pH.
Any material may be used as long as the reaction pH does not change up to the color change range of the indicator system. As a suitable buffering agent, a combination of one or more citrate buffering agents, fumaric acid buffering agents, tartaric acid buffering agents and the like can be preferably used.

Regarding the reaction conditions of 30 to 50 ° C., a heated sample is used, the reaction is carried out in an environment in which the temperature is preset, and when a test paper is used, the test is carried out on a heated plate. And the like.
When used as a test strip, a method of mixing a material that reacts with urine to generate heat is advantageous in terms of simplicity, but it may be placed near the test strip. As the material that reacts with urine to generate heat, materials that generate heat when dissolved, such as calcium chloride, calcium bromide, magnesium chloride, zinc bromide and zinc chloride, can be preferably used.

In the present invention, a solid-phase reagent prepared by dissolving the above-mentioned pH indicator, acidic buffer, and exothermic agent together with a polymer serving as a binder, impregnating an absorbent carrier or coating it on a carrier and drying it is preferably used. To do.

By using the solid phase reagent of the present invention, the total protein concentration in urine can be measured by the same operation as that for a normal urine test strip. In addition, by using together with a test strip for albumin measurement which does not contain a heating element, it is possible to measure γ-globulin and Bence Jones protein which are proteins other than albumin.

Example 1 Preparation of Test Piece for Measuring Protein in Urine First Stage Impregnation Solution Formulation Tetrabromophenol blue (TBPB) 50 mg Tetrabromophenolphthalein-ethyl ester potassium salt (TBPP-K) 8 mg 1N-sodium hydroxide Aqueous solution 450 μl Purified water 120 ml Second stage impregnation liquid formulation Citric acid / H ₂ O 15 g Trisodium citrate / 2H ₂ O 5 g Purified water 110 ml

Filter paper 3MMChr manufactured by Whatman is dipped in the first stage impregnating solution and dried, and then dipped in the second stage impregnating solution and dried. The obtained filter paper was cut into 5 mm squares and attached on a plastic strip.

Example 2 10 g of calcium chloride was added to the second-stage impregnation liquid in Example 1.
A test piece added with was prepared in the same manner as in Example 1. Urine known to contain only albumin as the protein and urine known to contain only Bence Jones protein were used as samples and measured using the test paper. The results are shown in Table 2.

[0016]

[Table 1]

The measuring device uses an integrating sphere type reflectance system, and a standard white plate of magnesium oxide has a reflectance of 100%. The samples used were urine, one prepared by adding albumin to urine, one prepared by adding γ-globulin to urine, and Bence Jones proteinuria. The reaction is 25 ℃
And on an aluminum plate whose temperature was adjusted to 37 ° C. In Figure 1,
The measurement results of urine containing only albumin, urine containing only globulin, urine containing albumin and globulin, and urine containing only Bence Jones protein at 25 ° C. and 37 ° C. are shown. The vertical axis represents the logarithmic conversion of the reciprocal of the reflectance R of the test piece, and the horizontal axis represents the protein concentration of the sample using the pyrogallol red method.

Example 3 Tables 1 and 2 show the temperature changes of the urine containing only albumin and the urine added to albumin urine so that the globulin content was 300 mg / dl using the reagent piece prepared in Example 1.

[0019]

[Table 1]

[0020]

[Table 2]

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[Procedure amendment]

[Submission date] September 2, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Added

[Correction content]

[Brief description of drawings]

FIG. 1 shows urine containing albumin alone, urine containing only γ-globulin, urine containing albumin and γ-globulin, and Bence Jones proteinuria at 25 ° C. and 37, respectively.
Measured with a urine test paper at ℃, logarithmically converted value of the reciprocal of the reflectance measured on the vertical axis, the protein concentration of the sample using the pyrogallol red method on the horizontal axis, plot all the measurement results It is a graph.

Claims (3)

[Claims] 1. A reaction temperature of 30 to 30 is used, which uses a dye that changes the color tone due to the presence of a buffer and a protein that impart acidic conditions.
A method for measuring protein in urine, which is performed at 50 ° C. 2. An absorbent carrier is impregnated with a buffering agent that imparts acidic conditions, a dye that changes color tone due to the presence of a protein, and a substance that generates heat due to the presence of an aqueous sample, or is formed on a support by coating. A measuring tool for urinary protein characterized by: 3. The device for measuring urinary protein according to claim 2, wherein the substance that generates heat due to the presence of the aqueous sample emits heat of dissolution.