JPH05164759A - Albumin measuring tool - Google Patents

Abstract

PURPOSE:To accurately detect an extremely small amount of albumin contained in urine by providing a fixed ligand which uniquely adsorbs albumin. CONSTITUTION:Urine penetrates into the title measuring tool from an injection port 3 and the albumin contained in the urine is adsorbed by a ligand fixed to a matrix 4. Then the urine is maintained in a water absorbing body 5 after the urine moves to and is absorbed by a water absorbing body 5 through the matrix 4. When the urine moves, the air contained in the matrix 4 and water absorbing body 5 is discharged to the outside through a communicative hole 6 and air channel 8. Therefore, the penetrating speed of the urine into the measuring tool is accelerated and albumin measurement can be carried out in a short time. In addition, the albumin measurement can be carried out without being affected by other proteins even when the amount of the albumin is extremely small. The concentration of a small amount of albumin can be precisely measured over an albumin concentration range of 0-20mg/dl.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring tool for measuring albumin in a sample. Specifically, the present invention relates to a measuring tool suitable for measuring a trace amount of albumin in a sample.

[0002]

2. Description of the Related Art Functional abnormalities of the kidney and bone marrow can be detected by examining proteins that appear in urine, and the screening method using a test strip is widely adopted in mass screening and various medical institutions because of its simplicity. There is.

In addition, diabetes has been increasing in recent years, and the occurrence of diabetic nephropathy, which is a complication of diabetes, is also increasing. It is said that diabetic nephropathy accounts for about one-third of vascular death in diabetes. However, early diagnosis is important. It has been recognized that the measurement of microalbumin contained in urine is effective for early diagnosis of diabetic nephropathy, and the measurement has been performed.

Generally, the upper limit of albumin in normal urine is set to 2.0 mg / dl. From this, in order to determine whether normal urine or abnormal urine is measured by measuring a trace amount of albumin, the measuring method is 2. It is necessary to be able to determine an amount of 0 mg / dl or less. However, the conventional screening method using test paper is not suitable for the measurement of trace albumin because its minimum detection sensitivity is 15 to 20 mg / dl.

To date, quantitative methods such as radioimmunoassay (RIA method), enzyme immunoassay method (EIA method), immunoturbidimetric method (TIA method), and latex agglutination inhibition method ( A semi-quantitative method such as a dye method [Ames; "Microbumin test" (trade name)] is known. RIA method, EI
Quantitative methods such as Method A and TIA can accurately measure the amount of microalbumin, but it takes a long time to measure, requires the use of special equipment and expensive reagents, and handles dangerous substances to the human body. It has the drawback that it must be used.

Semi-quantitative methods such as the latex agglutination inhibition method and the dye method can be performed more easily and in a shorter time than the quantitative method, and therefore, in the medical field, the above-mentioned RIA is performed.
These semi-quantitative methods are used as screening methods before performing the method, EIA method, TIA method, and the like. However, in the latex agglutination inhibition method, the detectable concentration of albumin is 3 mg / dl or more, and the above-mentioned 2.0 mg / dl
It cannot be used for the determination of trace albumin of dl or less, and the dye method has the drawback that it is difficult to determine the coloration in addition to being affected by proteins other than albumin [Clinical examination instrument reagent XII : 5 p945-951 (198)
9)].

[0007]

The present inventors have found that an extremely small amount of albumin in urine can be accurately detected by a simple operation and that the concentration range of albumin that can be measured is wide, and that there is no shortcoming as in the above-mentioned prior art, that is, an early stage. I have continued my research with the aim of obtaining an albumin measuring instrument that is useful for the diagnosis of diabetic nephropathy.

As a result, the inventors have found that the above object can be achieved by measuring albumin in a sample using a matrix on which a ligand that specifically adsorbs albumin is immobilized. That is, the present invention is an albumin measuring instrument comprising a matrix on which a ligand that specifically adsorbs albumin is immobilized.

To measure albumin in a sample using the albumin measuring instrument of the present invention, first, the sample (measurement sample) is passed through a matrix on which a ligand that specifically adsorbs albumin is immobilized. As a result, albumin in the sample is specifically adsorbed to the ligand,
Albumin is adsorbed and retained on the matrix in a concentrated state. Then, albumin adsorbed and retained in the matrix in a concentrated state is colored with a dye for albumin quantification, and the color intensity is examined visually or by using a device to measure the albumin concentration.

Incidentally, it is known that albumin is purified by using a matrix (for example, a fatty acid-immobilized column, blue sepharose etc.) on which a ligand capable of adsorbing albumin is immobilized [J. Biol. Chem. .2
48 (7), p2447-2451 (1973)], and in this case, because the purpose is to purify and recover albumin, it is a reversible ligand that can be eluted with an eluent after temporarily adsorbing albumin. It is necessary to use one. On the other hand, the present invention is not a method of purifying and recovering albumin, but an albumin measurement technique for measuring the presence or amount of albumin in a sample, which normally adsorbs albumin specifically and once The albumin thus formed does not necessarily have to be eluted again, so that the technical content is completely different from the above-mentioned albumin purification treatment.

In the present invention, it is preferable to use a ligand that specifically adsorbs albumin and does not cause a color reaction with a dye for measuring albumin. Any ligand having such properties can be used in the present invention, but as an example of a preferred ligand, the following formula (I);

[0012]

[Chemical 1] (In the formula, n represents a number of 10 to 20, particularly preferably 14 to 18).

The ligand represented by the above formula (I) specifically adsorbs albumin, does not cause a color reaction with an ordinary dye used for quantifying albumin, and further It has the excellent property of being adsorbed and held as it is without elution by the usual method.

The matrix on which the above-mentioned ligand that specifically adsorbs albumin is immobilized can be produced by binding the ligand to a carrier made of a liquid-permeable material. As the matrix to which the ligand represented by the above formula (I) is immobilized, for example, a liquid-permeable carrier having a hydroxyl group is used, and the hydroxyl group in the carrier has the following formula (II);

[0015]

[Chemical 2] It can be manufactured by reacting an epoxy compound represented by the formula (n represents a number of 10 to 20).

As a carrier for immobilizing a ligand, a sample can be passed through, and a ligand that specifically adsorbs albumin can be bound in a stable state, but it does not adsorb proteins and the like, and for measuring albumin. Any of those which do not cause a color-forming reaction with the above dye can be used. Preferred examples of the carrier that can be used in the present invention include filter paper, fleece of fibers having a hydroxyl group capable of reacting with the epoxy compound of the above formula (II), and fabrics such as knitted fabrics and nonwoven fabrics formed from the fibers, Inorganic and organic powders having a hydroxyl group, sponges and other porous materials can be mentioned.

The structure and shape of the measuring instrument of the present invention provided with the above-mentioned ligand-immobilized matrix is not limited as long as it can hold the matrix in a stable state and is easy to handle. The measuring tool having the structure shown in FIGS. 1 to 3 is particularly preferable because it has excellent albumin detection capability, sample permeability, and handleability. The measuring tool will be described below with reference to FIGS. 1 to 3.

FIG. 1 is a view from above of one of the preferred measuring tools of the present invention, and FIG. 2 shows the measuring tool of FIG.
It is a longitudinal cross-sectional view when cut along the line A. 1 and 2, 1 is a liquid-impermeable upper support, 2 is a liquid-impermeable lower support, 3 is a liquid sample (sample) inlet, and 4 is a ligand-immobilized matrix. .. Reference numeral 5 denotes a water absorbing body, and the amount of the liquid sample collected in the test device can be controlled by adjusting the type and size of the material. The water absorbing body is usually made of a water absorbing fiber such as cotton fiber or a cloth made of the fiber, a polymer water absorbing body, a paper product such as filter paper, a water absorbing inorganic material, a water absorbing sponge and the like. A communication hole 6 is provided between the water absorber 5 and the lower support 2.

As shown by the broken line in FIG. 1, the periphery of the upper support 1 is liquid-tightly coupled to the lower support 2 so that the injected liquid sample does not leak to the outside of the measuring tool.
Further, the lower support 2 is formed by two upper and lower long pieces closed at both side portions 7, and has an air channel 8 communicating with the central portion along the length direction thereof.

A method for measuring a sample, such as albumin in urine, using the above-mentioned measuring tool will be described. First,
The lower support 2 which plays the role of a handle is held by hand, and the upper support 1 of the measuring tool is immersed in urine for a predetermined time and then taken out. As a result, urine permeates the inside of the measuring tool through the inlet 3, and albumin contained in the urine is adsorbed by the ligand immobilized on the matrix 4. After that, the urine moves to the water absorbing body 5 through the matrix 4 and is absorbed and retained in the water absorbing body 5. As described above, the amount of the liquid sample collected in the test device, that is, the amount of albumin adsorbed to the ligand-immobilized matrix can be controlled by adjusting the type and size of the material of the water absorbent body 5. At that time, the air contained in the matrix 4 and the water absorbing body 5 is discharged to the outside of the measuring tool through the communication hole 6 and the air channel 8. Therefore, as compared with a closed albumin measuring instrument having no air channel, the rate of urine infiltration into the measuring instrument is accelerated, and albumin can be measured in a short time.

If the air channel 8 is too large, it becomes difficult to control the water absorption amount of the measuring tool. Therefore, it is preferable to adjust the size of the air channel 8 to control the water absorption amount within a predetermined range. Further, if the air channel 8 is too large, urine will be dripped from the air channel 8 when the measuring tool is inverted at the time of detection. From this point as well, it is necessary to adjust the size of the air channel 8. If necessary, a valve for preventing backflow or a water absorbing substance may be provided in the middle of the air channel 8 to prevent leakage of urine from the air channel 8.

The measuring tool of the present invention is not limited to the above, and, for example, in the measuring tool shown in FIG. 1 and FIG.
The lower support 2 which plays a role of promoting penetration of the handle and the specimen may be a hollow thin tube such as a straw or other shape. Moreover, for example, as shown in FIG.
It is also possible to provide a side peripheral wall 9 of a predetermined volume surrounding the periphery and measure a predetermined amount of the sample to be absorbed by the measuring tool at the side peripheral wall 9 portion.

Next, when a liquid containing a dye for measuring albumin was injected into the measuring instrument from the inlet 3 of the measuring instrument, it was proportional to the amount of albumin adsorbed and retained on the matrix 4, that is, the albumin concentration in urine. It shows the color intensity and can measure the albumin concentration in urine. Preferred examples of the albumin measuring dye that can be used in the measuring instrument of the present invention include tetrabromophenol blue, bromocresol green, bromocresol purple, tetrachloro-3,4,5,6-tetrabromosulfophthalein and the like. Examples include, but are not limited to: At that time, as the matrix 4, it is necessary to use a matrix that does not cause a color reaction with those dyes. The dye may be held in the matrix in advance, but if the dye is supplied to the measuring device after the albumin is adsorbed by the ligand immobilized on the matrix, a side reaction with a protein other than albumin will occur. It is desirable because it can measure the albumin concentration more accurately.

When the measuring tool as shown in FIGS. 1 to 3 is used, a portion (lower indicator) corresponding to the handle of the measuring tool is held by hand and the measuring tool is placed in the liquid sample for a predetermined time. Since it suffices to immerse the sample, it is possible to save the trouble of measuring a predetermined amount of the sample with a micropipette or the like and applying the sample to the injection port of the measuring tool, so that albumin can be measured more easily than in the past. Since the coloration state of the matrix changes sharply according to the albumin concentration in the sample, the determination can be performed with the naked eye, and if necessary, it can be performed using a measuring instrument.

With the above-mentioned measuring instrument of the present invention, the amount of albumin in the sample is about 2 mg / d, which corresponds to the amount of trace albumin.
It can be accurately and easily measured over a wide range of albumin concentrations from 1 to about 20 mg / dl with high albumin amount. The measuring instrument of the present invention is particularly suitable for detecting and measuring a minute amount of albumin in urine, but of course it can also be used for measuring a large amount of albumin in urine. Furthermore, the measuring device of the present invention can be used not only for albumin in urine but also for other liquids such as blood which require the measurement of albumin. For example, when the viscosity of the liquid to be tested is too high, the measurement can be easily performed by diluting it. become. Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto.

[0026]

【Example】

Example 1 (1) Synthesis of Ligand for Immobilizing Carrier: 0.1 mol of n-hexadecanol [CH ₃ (CH ₂ ) ₁₅ OH] was heated to 55 ° C. in a volumetric flask and then boron trifluoride. Ride ethyl ether complex (BF ₃ · Et ₂ O) is added 3 mmol. Next, 0.12 mmol of epichlorohydrin was added, and the mixture was reacted at 55 ° C. for 4 hours with vigorous stirring with a stirrer, and then left overnight at room temperature. Next, 50% N
10 g of an aOH aqueous solution is added and the reaction is carried out for 4 hours with stirring at room temperature. This reaction solution is transferred to a separating funnel by washing it with the same amount of water, further adding an appropriate amount of ether (about 30 ml), shaking well, and discarding the aqueous layer. Then, an appropriate amount of saturated saline is added and shaken well, the aqueous layer is stapled, the ether layer is transferred to an Erlenmeyer flask, 5 g of sodium sulfate is added and left overnight. Filtration to remove sodium sulphate and further ether removal by evaporation gives the formula (III):

[0027]

[Chemical 3] An epoxy compound represented by is obtained. The above-mentioned epoxy compound is synthesized by the following reaction formula.

[0028]

[Chemical 4]

(2) Production of matrix with immobilized ligand: A hydrophilic carrier having a hydroxyl group (here, filter paper: length × width = 7 cm × 24 cm) is wrapped with a net and immersed in 400 ml of dioxane. BF ₃・ E
Add 1 ml of t ₂ O and stir for 5 minutes. Next, 4 ml of the epoxy compound obtained in the above (1) and dioxane 4
0 ml was mixed with dioxane containing a filter paper carrier,
Add dropwise over 20 minutes with stirring. 40 after dropping
Continue stirring for minutes. This reaction is carried out in a nitrogen stream. As a result, the reaction of the epoxy group and the hydroxyl group causes the following formula (IV) on the filter paper carrier:

[0030]

[Chemical 5] A matrix in which the ligand represented by is immobilized on a filter paper carrier is obtained. The above-mentioned ligand-immobilized matrix was taken out, and 100% dioxane, 80%, 60%, 40%
And successively with 20% aqueous dioxane, and finally with water, and then air-dried at 60 ° C. for 50 minutes.

(3) Addition of buffer to matrix: The ligand-immobilized matrix prepared in (2) above was dipped in 0.1M KNa ₂ PO ₄ buffer (pH 7.5), taken out, and 60 ° C. Dry with air for 50 minutes.

(4) Preparation of albumin measuring device: width 11 mm, length 80 mm, thickness 0.
A hole (communication hole) having a size of 3 mm × 3 mm is provided in the vicinity of one end of a long piece made of a polystyrene sheet of 4 mm, and the porous hydrophilic polymer sheet acting as a water absorbing body so as to cover the hole ( (Made by Asahi Kasei; "Sunfine")
(10 mm × 10 mm) is placed on the ligand-immobilized matrix (10 mm × 10 m) obtained in (3) above.
m) (total thickness of filter paper and matrix is about 5 m)
m). On top of that, a piece of polystyrene sheet (10 m) as an upper support having a specimen injection port with a diameter of 4 mm in the center
m × 10 mm) and its periphery is liquid-tightly bonded to the lower support by an adhesive or heat fusion. Next, on the lower surface side of the polystyrene strip as a lower support, a width of 11 m
m, a length of 80 mm, and a thickness of 0.4 mm, which are made of polystyrene sheets, are overlapped, and both sides of both the polystyrene length pieces in the length direction and the side portion on which the matrix is placed have a thickness of 0. A polystyrene wall member having a width of 4 mm and a width of 3 mm is interposed and joined to form a handle portion having an air channel, and the measuring tool shown in FIGS. 1 and 2 is manufactured.

(5) Measurement of albumin in urine: (i) health
Commercial albumin standard in human urine ("Human Albu" manufactured by Sigma)
min ”) is added to test different albumin concentrations.
Multiple specimens (urine) were prepared. (4) in each test urine
The measuring tool prepared in 1. was dipped for 30 seconds and then taken out.
Next, inject one drop of the coloring liquid having the following composition into the measuring tool.
It was dripped at the inlet.Coloring liquid composition  Tetra bromphenol blue 0.36
mM Nikkor BB-20 (Nikko Chemicals) 0.1% citrate buffer (pH 3.2) 0.5 M
The resulting color intensity is reflected at a wavelength of 620 nm.
As the absorbance, a reflection absorptiometer (manufactured by Otsuka Electronics Co., Ltd .; MCP
D-200).

As a result, as shown in FIG. 4, the albumin concentration in urine and the reflection absorbance at 620 nm in the measuring device have a substantially proportional relationship over a range of albumin concentration of about 2 to 20 mg / dl. From this, when the measuring device of the present invention is used, the albumin concentration is about 2 to
It is clear that albumin in urine can be measured almost accurately over a wide range of 20 mg / dl.

Further, when the color intensity which appeared on the measuring tool was visually judged at the same time using the standard color tone table, it was found to be 0.
~ 2 mg / dl, 3-7 mg / dl, 8-15 mg / d
It was possible to judge the albumin concentration by dividing it into 4 steps of 1 and 16 mg / dl or more. From this, it can be seen that the test device of the present invention can be effectively used for visual measurement of albumin concentration in urine, particularly for measurement of trace amount of albumin having a concentration of 3 mg / dl or more.

(Ii) Next, 25 urine samples of actual diabetic patients were measured using the above-mentioned measuring tools. The reflection absorbance at a wavelength of 620 nm of the color intensity appearing in the measuring tool was measured in the same manner as above. At the same time, Dainippon Pharmaceutical Co., Ltd.
The albumin concentration of each urine was precisely measured by EIA method using "Albumin M-EIA" manufactured by. For each urine, the measurement result of albumin concentration by the EIA method was plotted on the horizontal axis, and the reflection absorbance at 620 nm by the measuring tool of the present invention was plotted on the vertical axis. The result is as shown in the graph of FIG. Based on the result of FIG.
When the correlation between the result of the precision measurement by the method A and the result by the measuring tool of the present invention was examined, the albumin concentration was 0 to 8
In the range of mg / dl, a good correlation with a correlation coefficient γ = 0.88 obtained by the following mathematical formula 1 was shown. The overall correlation coefficient was γ = 0.91, indicating a very good correlation.

[0037]

[Equation 1] (In the formula, Sx represents the standard deviation of x, and Sy represents the standard deviation of y.)

(Iii) Furthermore, using the above-mentioned measuring tool,
Twenty-five actual human urine samples were measured. The color intensity that appeared in the measuring tool was measured as the reflection absorbance at 620 nm in the same manner as above. At the same time, the total protein concentration of each urine was measured using "Protein Assay" manufactured by Bio-Rad. For each urine, the measurement result of the total protein concentration was plotted on the abscissa, and the reflection absorbance at 620 nm by the measuring tool was plotted on the ordinate. As a result, the graph shown in FIG. 6 was obtained. Based on the results of FIG. 6, the correlation with the results of the measuring device of the present invention was examined in the same manner as in (ii) above, and when the total protein concentration was in the range of about 0 to 20 mg / dl,
The correlation coefficient was γ = 0.44, which was an extremely low value.

From the results of (ii) and (iii) above, the albumin concentration was about 0-8 mg / dl and the total protein concentration was about 0-2.
In the low concentration range of 0 mg / dl, the measuring instrument of the present invention shows an extremely high correlation coefficient with the albumin concentration, whereas it has an extremely low correlation coefficient with the total protein concentration. Therefore, it can be understood that the measuring instrument of the present invention specifically adsorbs albumin and can measure the concentration of albumin almost accurately without being affected by other proteins contained in urine.

[0040]

INDUSTRIAL APPLICABILITY The albumin concentration measuring instrument of the present invention specifically adsorbs albumin and can accurately measure albumin concentration in a sample without being interfered by other proteins. It is possible to measure. Therefore, 3 mg / is important for the diagnosis of early diabetic nephropathy.
The albumin concentration around dl can be measured, and the early diabetic nephropathy can be easily and accurately diagnosed.

Furthermore, when the measuring instrument of the present invention is used, the color intensity and the reflection absorbance of the measuring instrument change largely and clearly over the albumin concentration range of 0 to 20 mg / dl. Each trace albumin concentration can be measured accurately. Moreover, in the case of the measuring instrument of the present invention, it is possible to measure albumin concentration in a sample in a short time with an extremely simple operation, without using complicated operations, expensive devices and reagents, dangerous reagents and the like. it can.

[Brief description of drawings]

FIG. 1 is a view showing an example of an albumin concentration measuring instrument of the present invention, which is a view of the measuring instrument seen from above.

FIG. 2 is a vertical sectional view of the measuring tool of FIG. 1 taken along the cutting line AA.

FIG. 3 is a diagram showing another example of the albumin concentration measuring device of the present invention.

FIG. 4 is a diagram showing a relationship between reflection absorbance at a wavelength of 620 nm and albumin concentration in healthy urine to which different amounts of albumin preparations were added when examined by using the measuring instrument of the present invention. is there.

FIG. 5 is a graph plotting the relationship between the reflection absorbance at a wavelength of 620 nm when the albumin concentration in an actual human urine sample is examined by the measuring device of the present invention, and the albumin concentration by the EIA method.

FIG. 6: Reflection absorbance at a wavelength of 620 nm when the total protein concentration in an actual human urine sample was examined by the measuring instrument of the present invention, and “Protein Assay” manufactured by Bio-Rad Co.
It is the figure which plotted the relationship with the total protein concentration by.

[Explanation of symbols]

 1 Upper support 2 Lower support 3 Specimen injection port 4 Ligand immobilization matrix 5 Water absorber 6 Communication hole 7 Lower support side 8 Air channel 9 Side wall

Claims (1)

[Claims] 1. An albumin measuring instrument comprising a matrix on which a ligand that specifically adsorbs albumin is immobilized.