JPH0635974B2 - Test piece for protein measurement - Google Patents

Description

TECHNICAL FIELD The present invention relates to a test strip for protein measurement, which is used for simple measurement of protein in body fluids such as urine, serum, plasma, and cerebrospinal fluid, especially urine.

[Background of the Invention]

It is said that even a healthy person excretes 20 to 80 mg of protein in urine every day, but the excreted protein is mainly albumin whose particles are small and easily pass through the glomerulus.
On the other hand, hemoglobin is heavily hemolyzed and hemoglobin of red blood cells migrates into plasma in large amounts, which leaks from the renal glomerulus, or when there is inflammation in the kidney or urinary tract, it releases white blood cells into the urine. It becomes urine protein that does. Urine protein generally has a high value in the following cases and is an important guideline for renal diseases.

(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) Obstruction of the ureter due to stenosis, stones, or tumor.

(7) Brain tumor, epilepsy, other central nervous system diseases, emotional movement.

(8) Mixture of pus, blood, semen, etc.

(9) Ingestion of large amounts of proteins with small molecular weight such as eggs.

(10) A temporary thing that appears after intense exercise, prolonged use in hot or cold water.

(11) Postural and juvenile proteinuria.

At present, the following methods are commonly used for measuring urine protein.

(1) Sulfosalicylic acid method (sensitivity 0.002%) Clear urine 4-5 ml + sulfosalicylic acid 20 W / V% solution 2-
3 drops → white If turbid or precipitated, protein positive.

(2) Boiling test method (sensitivity: about 0.005%) 5 ml of clear urine is boiled for 1 to 2 minutes, and if turbidity occurs, add 1 to 3 drops of 5% acetic acid or 70% nitric acid when hot. Protein is positive if unchanged or increased.

(3) Robers method (sensitivity 0.003%) If the sample and the nitric acid solution of magnesium sulfate are mixed in equal volumes and a white ring is not formed on the interface, protein is positive.

(4) Test paper method (sensitivity 0.03%) A protein error method is used in which the pH indicator such as bromphenol blue or tetrabromphenol blue produces a color on the alkaline side due to the presence of protein.

(5) Coomassie brilliant blue method (sensitivity 0.001%) It is a highly sensitive measurement method by binding of dye and protein.
l + CBB-G250 solution 3 ml → Measure the absorbance at 595 nm.

(6) Biuret method by trichloroacetic acid precipitation Sample (original urine) 2 ml + distilled water 2 ml + trichloroacetic acid (10
% Solution) After mixing, centrifuge at 3,000 rpm for 5 minutes or longer and discard the supernatant. 2 ml of Biuret's reagent (NaOH 4%, potassium sodium tartrate crystal 4.5%, CuSO ₄ .5H ₂ O 0.5%, potassium iodide 0.5%) + distilled water 2 ml was added to the precipitate, and the mixture was heated at 37 ° C. for 30 minutes and colorimetrically measured at 540 nm.

(7) Pyrogallol red method This is a high-sensitivity measurement method that utilizes the metachromatism phenomenon of proteins, and the sample + acid solution of pyrogallol red and molybdic acid red complex → the absorbance at 600 nm is measured.

Among these, sulfosalicylic acid method, boiling test method, Robers
The method requires a large amount of sample by the nephelometric method or a method similar thereto, and has a limit in accuracy when applied to quantitative analysis. On the other hand, the Coomassie Brilliant Blue method, which is a colorimetric method, is considered unsuitable for continuous treatment of many samples because of the curved calibration curve and contamination of cells, test tubes, etc.

Further, the biuret method by trichloroacetic acid precipitation requires a precipitation separation operation, and therefore the operation is complicated and not practical.

The pyrogallol red method is a method that can measure trace proteins in a sample with high sensitivity and is widely used, but since it is a colorimetric method and requires time for measurement, it can be easily used for purposes such as screening. It is hard to say that it is a method.

Moreover, the test strip method utilizing the protein error method is very simple and widely used for purposes such as screening tests. However, all of these test strips currently on the market are, for example, exposed by the naked eye. At the time of comparison and judgment with the color sample, there were problems such as difficulty in judgment in the boundary area between normal and abnormal and low measurement sensitivity.

Therefore, as the measurement of urinary protein is attracting attention as a good indicator for early detection of various renal diseases, there is an increasing demand for a simple, sensitive, and accurate measurement method for urinary protein. .

[Object of the Invention]

The present invention has been made in view of the current situation as described above, and an object of the present invention is to provide a simple, highly sensitive and highly accurate protein measuring method by a test strip method.

[Structure of Invention]

The present invention relates to a test piece for protein measurement in which a support for protein measurement is impregnated on a support and dried, and an absorbent carrier is present in the test piece for protein measurement. The test piece for protein measurement is characterized in that the carrier is previously calendered.

That is, the inventors of the present invention have been earnestly researching in order to develop a test piece for protein measurement, which is simple, has high sensitivity, and has high accuracy, and is preliminarily calendered as an absorbent carrier impregnated with and dried with reagents for protein measurement. The inventors have found that the above objects can be achieved by using the above-mentioned products, and completed the present invention.

The test piece for protein measurement of the present invention is prepared by impregnating the reagents for measuring protein, and an absorbent carrier to be dried, except that calender treatment is carried out in advance, according to a method for preparing a test piece for protein measurement known per se. Can be prepared. That is, the absorbent carrier that has been subjected to a calendar treatment in advance is impregnated with the reagents for protein measurement in 1 to several times by a conventional method and dried. The test piece for protein measurement of the present invention can be obtained by cutting this into an appropriate size and immobilizing it on an appropriate support by a conventional method using a double-sided adhesive tape or the like.

The calendar processing referred to in the present invention is a so-called "calende (calende)" which is generally performed as a finishing treatment for paper or cloth.
r) ”treatment, which is a rolling treatment that smoothes the surface of paper or cloth, gives light selection, and in the case of papers, makes the paper quality dense.

In the test piece for protein measurement of the present invention, as the absorbent carrier used in advance by calendering, paper, cellulose,
Absorbent carriers used in ordinary test pieces for measurement, such as chemical fibers, synthetic resin woven fabrics, and non-woven fabrics, are all examples without exception. These absorptive carriers are not necessarily limited, but the thickness after impregnating the reagents for protein measurement and drying is preferably 0.25 mm or less.

Further, for example, when using paper as the absorbent carrier, the calendering can be carried out continuously with the paper making step, or can be carried out as another step, or both, and the finishing method used for calendering is also possible. Any method commonly used in the paper industry such as plate calender and super calender may be used without any particular limitation.

Further, when paper is used as the absorptive carrier, it is preferable to increase the beating degree in the beating treatment in the steps performed in the paper making step, as compared with the case where ordinary chromatographic paper is used. The beating process is not particularly limited as long as it is usually performed in the paper industry.

Reagents for protein measurement used in the present invention do not need to be particularly selected for the present invention, and a protein detection dye used in an existing protein measurement test strip,
A buffering agent or the like can be used without any trouble. That is,
The dye for protein detection can be used without particular limitation as long as it is a dye that causes metachromatism due to protein, for example, tetrabromophenol blue, tetrabromophenolphthalein ethyl ester, congo red, bromcresol green. Usually, methyl orange, a complex of pyrogallol red and molybdic acid, a complex of pyrocatechol violet and molybdic acid, etc. are preferably used. Use these alone,
Alternatively, it is optional to use two or more kinds in combination. Also,
The amount of these dyes used is usually the concentration of the impregnating liquid.
A preferable range is 0.001 to 1%. The operation of impregnating the impregnating liquid into the absorbent carrier and drying may be carried out by a conventional method, and the operation of impregnation / drying may be performed once, or may be repeated several times.

In addition, as the buffering agent, a buffering agent that can normally be used on the acidic side, for example, a citrate buffering solution, a glycine-hydrochloric acid buffering solution and the like can be mentioned as a typical one. Regarding, since the preferred ones themselves differ depending on the dye used, depending on the dye used,
It goes without saying that the buffering agent to be used and its concentration should be appropriately selected and used.

These reagents for measuring proteins are dissolved in water, a suitable organic solvent or a mixed solvent thereof as an impregnating solution, and the absorbent carrier according to the present invention is immersed in the impregnating solution to absorb the reagents. Impregnate the carrier.

As the support for holding the absorbent carrier according to the present invention,
All existing test strips for protein measurement can be used without exception as long as they are usually used as a support, and examples thereof include glass fiber, polyvinyl chloride, Teflon, polystyrene, polyvinyl acetal, acetyl cellulose, nitrocellulose, Examples thereof include sheets of synthetic polymer compounds such as polyvinylidene chloride and polypropylene, and cardboard coated with these. Among them, those having the property of easily reflecting light themselves, or making them easy to reflect light A processed product, for example, a white polymer film with light selection or a polymer film with an aluminum foil with light selection attached is more preferably selected. Further, instead of using a support having such a property, even if a property that easily reflects light between the absorptive carrier and the support, for example, an aluminum foil having a selection is provided, the same effect can be obtained. As a result, when such a treatment is carried out, the range of selection of the support is further expanded.

There are no particular restrictions on the sizes and dimensions of these absorbent carriers and supports, and those that conform to the sizes and dimensions of the absorbent carriers and supports in commonly used measurement test pieces should be used. Is enough.

In addition, regarding the method of adhering the absorbent carrier to the support, any method that is usually used may be used when preparing a test piece for protein measurement, such as a method using an adhesive and a method using an adhesive tape. I don't need any special method,
It is more preferable that the adhesive or the adhesive tape itself is transparent, or that the adhesive or the adhesive tape itself easily reflects light.

In the conventional test piece for protein measurement, by using a thicker absorbent carrier as a material than the test pieces of other measurement items, and increasing the amount of the sample impregnated in the absorbent carrier,
The measurement sensitivity was improved. Therefore, it was surprising that the measurement sensitivity can be improved by using the absorptive carrier which has been calendered to be thinner than the conventional one as in the present invention.

The reason for this is not clear yet, but by using the calendered absorbent carrier as a material,
The surface of the finished test piece was smoothed, and the decrease in reflectance due to diffuse reflection was prevented, or when the finished test piece was impregnated with a sample, when using a conventional test piece Compared with the above, the transparency of the test piece itself is increased, and the light passing through the test piece is more likely to be reflected by the support and returned to the surface of the test piece. It can be considered that the change in is easier to determine.

Examples will be shown below, but the present invention is not limited to these examples.

〔Example〕

Example 1. (1) Sample urine Human albumin (manufactured by Sigma) was added to human urine pooled with protein albumin which was determined to be protein-negative by a commercially available test strip for protein measurement in an amount of 5,10,1.
Samples were added at 5, 20, 30, 100, 300 and 1000 mg / dl.

(2) Preparation of Impregnation Solution (Solution A) Citric acid 8.1 g Sodium citrate 3.5 g These were dissolved in 40 ml of distilled water and adjusted to pH 3.1 with a 1.5 M citric acid solution to a total volume of 50 ml.

(Solution B) Tetrabromophenol blue 60 mg This was dissolved in methanol to make 50 ml.

Then, the above liquids A and B were mixed to prepare an impregnating liquid.

(3) Preparation of test piece Calendered chromatograph paper (thickness 0.18 mm)
Was impregnated with the above impregnating solution by a conventional method and dried. The test paper prepared in this way is cut into squares of 5 mm square,
Polyvinyl chloride sheet (5mm × 8c) with double-sided tape
The test piece was attached to m).

(4) Measurement method and measurement results After each test piece was dipped into each sample urine and impregnated with the test piece, the degree of coloration was measured after about 30 seconds. The degree of coloration was measured as a reflectance at a measurement wavelength of 635 nm using a urinalysis instrument. The results are shown in Table 1.

Comparative Example 1. Instead of the calendered chromatographic paper in Example 1, a 0.28 mm-thick uncalendered chromatographic paper was used, and the same impregnating solution as in Example 1 was used. A test piece was prepared by the method described in (1), and the same sample urine as in Example 1 was measured in the same manner as in Example 1.
The results are also shown in Table 1.

As is clear from Table 1, in the test piece of the present invention, the reflectance difference in the vicinity of the boundary between the normal range and the abnormal range (human serum albumin concentration 10 to 30 mg / dl) is higher than that in the conventional test piece. You can see that is getting bigger. That is, it can be seen that the color change near the boundary between the normal region and the abnormal region, which was difficult to discriminate with the conventional test piece, became clear.

Example 2. (1) Sample urine The same sample urine as in Example 1 was used.

(2) Preparation of Impregnating Solution The same impregnating solution as in Example 1 was used.

(3) Preparation of test piece A test piece was prepared in the same manner as in Example 1.

(4) Measurement method and measurement results The test pieces were instantaneously dipped in the sample urine to impregnate the test pieces, and the coloration of the test pieces was observed after about 10 seconds. The test paper part was colored yellow to blue depending on the albumin concentration of the sample.
The detection limit was 5 mg / dl.

Comparative Example 2. In place of the calendered chromatographic paper in Example 2, a non-calendarized chromatographic paper having a thickness of 0.4 mm was used, and the same impregnating solution as in Example 2 was used. A test piece was prepared by the method described in 1 above, and the same sample urine as in Example 2 was measured in the same manner as in Example 2.

The test paper portion of the test piece was colored yellow to greenish blue depending on the albumin concentration of each sample. Visual detection limit is about 10
It was mg / dl.

Example 3. (1) Sample urine The same sample urine as in Example 1 was used.

(2) Preparation of Impregnation Solution (Solution A) Citric acid 8.1 g Sodium citrate 3.5 g These were dissolved in 50 ml of distilled water and adjusted to pH 3.0 with a 1.5 M citric acid solution to a total volume of 50 ml.

(Solution B) Tetrabromophenol phthalein ethyl ester 50 mg This was dissolved in methanol to make 50 ml.

Then, the above liquids A and B were mixed to prepare an impregnating liquid.

(2) Preparation of test piece A test piece was prepared in the same manner as in Example 2.

(3) Measurement method and measurement result The measurement was performed in the same manner as in Example 2. The test paper portion was colored yellow to blue depending on the albumin concentration of the sample. The limit of visual detection was 5 mg / dl.

When the changes in coloration of the test pieces of Examples 2 and 3 and Comparative Example 2 were compared, that of Examples 2 and 3 had a brighter overall color tone than that of Comparative Example 2, particularly in the normal range and abnormal range. The change in coloration near the boundary at was more visible.

〔The invention's effect〕

As described above, the present invention provides a simple, sensitive and highly accurate protein measuring method by a test strip method, which is difficult to discriminate with a conventional protein measuring test piece, and a normal range and an abnormal range. This is an invention that has a remarkable effect in that the distinction in the vicinity of the boundary becomes clear, and is an extremely large invention that contributes to the related art.

Claims (5)

[Claims] 1. A support is impregnated with reagents for measuring protein,
In a test strip for protein measurement in which a dried absorbent carrier is present, the absorbent carrier to be impregnated with the reagents for protein measurement and dried is calendered in advance. Test piece. 2. The test piece according to claim 1, wherein the absorbent carrier impregnated with the reagents for measuring protein and dried has a thickness of 0.25 mm or less. 3. The test piece according to claim 1, wherein the support is made of a material that easily reflects light or processed so as to easily reflect light. 4. The test piece according to claim 1, wherein a substance having a property of easily reflecting light is present between the absorbent carrier and the support, which are impregnated with the reagents for measuring proteins and dried. . 5. The test piece according to claim 1, wherein the protein is urinary protein.