JPS60147651A - Test paper for detecting total protein and amino acid in body fluid and its production - Google Patents

Abstract

PURPOSE:To enable detection of the protein and amino acids contained in a specimen soln. with high accuracy by impregnating an alkaline buffer soln. contg. trinitrobenzenesulfonic acid with an absorptive carrier. CONSTITUTION:Trinitrobenzenesulfonic acid (TNBS) is dissolved in an alkaline buffer soln. of, for example, potassium dihydrogenphosphate or the like and an absorptive carrier of a hydrophilic high polymer carrier such as filter paper or non-woven fabric is dipped in the soln. and is dried after impregnation to remove thoroughly the moisture, thereby manufacturing test paper. When the test paper is dipped in a specimen soln., for example, unine or the like, the amino group possessed by protein reacts with TNBS and forms a color and therefore not only the protein in the urine but also the compd. having the amino acid and amino group in the specimen are detected. Detection of all kinds of protein is thus made possible and the thorough checking of the protein in the urine inspection is accomplished.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field The present invention relates to a test strip for detecting protein in body fluids, particularly urine protein, and a method for producing the same. More specifically, the present invention relates to a test strip capable of detecting total protein contained in a sample solution, and a method for producing the same.

BACKGROUND OF THE INVENTION A simple method for detecting urinary protein, instead of the conventional precipitation method, is a color reaction using a test strip.

Japanese Patent Publication No. 43-7991 discloses a method for producing a test paper in which an absorbent carrier is impregnated with a buffer solution, dried, and then impregnated with a dye dissolved in a halogenated hydrocarbon. Using the test strip obtained by this method, the protein level is 0.0
Detected with a detection limit of 0.05%.

However, components other than protein contained in urine may also give a weak positive result, and albumin is the only protein detected by this test strip.

Proteinuria usually only contains albumin, so only albumin needs to be detected.

However, in some cases of hyperproteinuria, the urine contains T-globulin rather than albumin.

Since T-globulin is not detected by this test strip, the disease may be overlooked. Special Public Service 1977-18
Publication No. 73 discloses a method for producing a test paper in which an absorbent carrier is loaded with a dye showing a protein error and a buffer solution, and the carrier is impregnated with an inorganic sulfate.

The detection limits for proteins using test strips manufactured using this method are at the same high level of <0.005%.
Moreover, false positive reactions due to components other than proteins do not occur.

However, the protein detected by this test strip is only albumin, and T-globulin is not detected.

Amino acids, amino sugars, proteins, etc. undergo the following substitution reaction with trinitrobenzenesulfonic acid (TNBS), and the product has an absorption near 420 nm.
J, Biochem, + by KuyalIla et al.
47°454 (1960).

■: It would be convenient if this method, such as peptide chains, could be applied to test strips for detecting proteins contained in specimens. However, (1) T
NBS is unstable under alkaline conditions, so it is used for qualitative or quantitative determination of proteins.

There are problems such as: it is necessary to mix the alkaline buffer and TNBS solution with the specimen sample during measurement; and (2) it is necessary to heat it to about 70° C. to promote the reaction.

OBJECTS OF THE INVENTION An object of the present invention is to provide a test strip that can detect proteins and amino acids contained in a sample solution with high precision, and a method for producing the same. Another object of the present invention is to provide a test strip that can detect all kinds of proteins contained in a sample solution and a method for manufacturing the same.

Summary of the Invention The test strip of the present invention is an application of the principle of the method of Okuyama et al., and is obtained by impregnating an absorbent carrier with an alkaline buffer solution containing TNBS, thereby achieving the above object. be done. Furthermore, the method for producing a test strip of the present invention includes a step of impregnating an absorbent carrier with an alkaline buffer solution containing TNBS, thereby achieving the above object.

The alkaline buffer used in the present invention has a pH of 8 to 10.
This solution has a buffering capacity in the range of . Potassium dihydrogen phosphate-sodium hydroxide.

Boric acid - Potassium chloride - Sodium hydroxide, Sodium tetraborate - Sodium hydroxide, Potassium dihydrogen phosphate -
Disodium hydrogen phosphate, potassium dihydrogen phosphate-sodium tetraborate, sodium tetraborate-sodium carbonate, boric acid-potassium chloride-sodium carbonate, boric acid-sodium chloride-sodium tetraborate, sodium carbonate-
Sodium hydrogen carbonate and the like are used. Furthermore, Br1t
ton-Robinson's broad spectrum buffer and the like may also be used. Among these buffers, a buffer containing sodium tetraborate-potassium dihydrogen phosphate is preferred in terms of its stability. The ionic strength of these buffers is 10-5
00mM and pH is 8-10. When pt+ is 8 or less, color development at room temperature is extremely slow. When pl+ is 10 or more, TNBS decomposes in the drying process when mixed with TNBS and impregnated into a test paper. As a result, the test paper itself becomes colored. However, the higher the pH, the higher the degree of color development. The optimum p-region is 8.3 to 9.3. TNB
S is contained in the buffer at a rate of 0.3 to 10 mg/mit. If the TNBS content is 0.3 mg/m2 or less, the degree of color development will be low, and if it is 10 mg/mβ or more, the base will develop color in the process of impregnating with the TNBS-containing solution and drying, making it unusable.

Absorbent carriers that can be used in the method of the invention include, for example, filter paper and hydrophilic polymeric carriers such as crosslinked popal and nonwoven fabrics.

The target urine protein test strip can be obtained by dissolving TNBS in the above buffer solution and immersing the above absorbent carrier in the solution.

The buffer solution and the TNBS solution may be prepared separately, and the absorbent carrier may be impregnated with these in sequence. From the viewpoint of the stability of the test strip, it is desirable that the absorbent carrier impregnated with TNBS and the buffer coat completely removes moisture during drying. For this purpose, the impregnated absorbent carrier is preferably lyophilized. It is advisable to immediately transfer the dried test strip to a brown bottle containing a desiccant or a container that can block moisture and light for storage.

Protein can be detected by immersing the obtained test strip in a sample solution such as urine. Since the amino groups of proteins react with TNBS to produce color, it is effective for detecting not only proteins in urine but also amino acids contained in samples and compounds with amino groups such as amino sugars. When performing a urine protein detection test using this test paper, the test paper is used as is, or a small piece of the test paper is fixed to the end of a polymer stick. After immersing this test piece in the test urine.

Take it out immediately and remove excess urine with filter paper etc.
Color development stabilizes in 0 to 80 seconds. This test paper develops an orange-yellow color depending on the concentration of compounds with amino groups contained in the urine sample. This allows the protein to be detected. This method is particularly effective in cases where proteins other than albumin appear in the urine, such as in diabetic nephropathy. Furthermore, if a standard colorimetric table is prepared under certain conditions, it is possible to quantify the protein contained, and it functions as a semi-quantitative test paper.

Example The present invention will be described below with reference to Examples.

Example 1 TNBS was added to a mixture of 2 parts by volume of 0.1M potassium dihydrogen phosphate and 8 parts by volume of 0.05M sodium tetraborate.
It was added to a concentration of 0 mg/dβ. Toyo Roshi N12 (8 cm x 20 cm) was immersed in this solution to impregnate it. After impregnation, it was immediately freeze-dried and cut into small pieces of 5 m x 5 cranes. A polymer stick was prepared by cutting a polystyrene film with a thickness of 300 μm into a size of 50 n×5 m, and the above test paper piece was fixed to the tip of the polymer stain with double-sided tape. Separately human albumin, α-
Globulin, β-globulin and T-globulin 3
Standard solutions adjusted to concentrations of 0.50, 100°250, and 1000 mg/dIl were prepared. After the test paper was immersed in each standard solution, it was immediately pulled out, excess moisture was removed with a filter paper, and the test paper was left for 60 seconds. The developed color tone was read using a standard colorimetric table. The results are shown in the table below.

As shown in the table, the test paper according to the method of the present invention is 0.0
It was found that it was possible to detect proteins at a concentration of 3% and had sufficient sensitivity for various proteins as a qualitative test paper for total protein.

Effects of the Invention According to the present invention, it is possible to provide a test strip that can detect proteins contained in a sample solution such as urine with high accuracy regardless of the type thereof.

Using this test strip, it is possible to detect all kinds of proteins, including T-globulin, which could not be detected conventionally, so that proteins can be completely checked in urine tests.

that's all Agent: Patent Attorney Shusaku Yamamoto

Claims (1)

[Scope of Claims] 1. A test strip for detecting proteins and amino acids in body fluids impregnated with an alkaline buffer solution containing I-'J nitrobenzene sulfone m. 2. A method for producing a test strip for detecting proteins and amino acids in body fluids, which comprises impregnating an absorbent carrier with an alkaline buffer solution containing trinitrobenzenesulfonic acid.