JPH11304803A - Analysis method of protein in sample and kit thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce the number of processes as much as possible and perform a color measurement in a short time by mixing a pigment which forms an ion associated body with protein to a sample, and making the formed ion associated body adsorbed by a hydrophobic filter. SOLUTION: First, a hydrophobic filter 2 is fixed to the opening part 4 of a cylinder 3 without gaps. An ion associated body solution obtained by mixing a sample with a pigment is poured in advance into a cylinder 3, a piston 5 is moved in the direction of the opening part 4 (or a lever 6 is pressed), and the ion associated body solution is pushed out of the cylinder 3. At this time, the ion associated body included in the ion associated body solution is adsorbed by the hydrophobic filter 2. Alternatively, the ion associated body solution may be sucked into the cylinder 3 so as to make the hydrophobic filter 2 adsorb the ion associated body, by introducing the opening part 4 into the ion associated body solution obtained through the addition of the pigment to the sample and moving the piston 5 on the opposite side to the opening part 4 (or pulling the lever 6).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an analysis method and an analysis kit which can easily and qualitatively and quantitatively determine a minute amount of a protein contained in a sample. It is useful in the fields of analytical chemistry, life science, and medicine, especially in the field of clinical testing.

[0002]

2. Description of the Related Art Conventionally, the simplest means for protein analysis is a test piece in which a carrier is impregnated with a dye that changes its color tone when bound to a protein. However, this test piece can absorb only a few μl to several tens μl of a sample, and it is difficult to apply a sample containing only a small amount of protein. In addition, other substances and unbound dyes in the sample may be affected, resulting in erroneous results.

[0003] For this purpose, in recent years, several techniques have been proposed for concentrating a sample to increase the sensitivity and reducing the influence of interfering substances in the sample.

For example, Kinoshita et al. [Analytical Chemistry 23, 1543-1]
544, 1974] report a technique in which a protein in a sample is concentrated on a membrane filter, the filter is then stained with fluorescein isothiocyanate (FITC), and after washing, the fluorescence intensity of the spot is measured.
However, in this technique, protein and FI
There was a problem that the reaction with TC required as long as 30 minutes and that washing was required after staining to remove interfering substances.

Also, Fujita et al. [Analytical Chemistry 44, 733-738]
Pp. 1995] reported a technique for physically collecting a precipitated aggregate of a protein and a dye-metal complex with a filter, re-dissolving with an aqueous sodium hydroxide solution, and measuring the absorbance of the solution. I have. However, in this technique, the quantitativeness is not always good due to clogging of the precipitate, and the coloring on the filter is not observed,
Since the re-dissolved solution was measured, it was not suitable for simple and rapid analysis.

[0006]

As described above, the conventional techniques for increasing the sensitivity by concentrating a sample containing only a small amount of protein, such as the analysis of protein in urine, include a washing step, a coloring step, and an extracting step. Since the step (1) is indispensable, the operation is complicated, it takes time to measure the color, and there is a problem in the quantitativeness. Therefore, an object of the present invention is to provide a simple and rapid protein analysis means which minimizes the number of steps, enables color measurement in a short time, and has excellent quantitative properties.

[0007]

According to the present invention, there is provided a method comprising the steps of: a) mixing a sample which may contain a protein with a dye which forms an ion aggregate with the protein, and precipitating the ion aggregate without precipitating the ion aggregate; Forming a combined solution; b) passing the ion-associated solution through a hydrophobic filter so that the aggregate dissolved in the ion-associated solution is adsorbed to the filter; and c) on the filter. A method for analyzing a protein in a sample, wherein the step of qualitatively and quantitatively analyzing the above aggregate is sequentially performed.

[0008] The present invention also relates to a protein used in the above method, which comprises a dye for forming an ion associate with the protein and a hydrophobic filter, and optionally a device for passing the ion associate solution through the hydrophobic filter. This is an analysis kit. As an apparatus for passing the ion association solution through a hydrophobic filter, for example, an opening capable of fixing the filter can be moved in the direction of the one end by sliding the cylinder at one end thereof and the inner wall of the cylinder. And an externally operable piston. The movement of the piston causes the ion association in the ion association solution to be adsorbed to the filter.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Samples that can be analyzed in the present invention include, for example, biological samples (blood, serum, plasma, urine, cerebrospinal fluid, semen, body fluids such as saliva, sweat, ascites, amniotic fluid, and tears), brain, and liver. And extracts of organs such as kidneys, extracts of tissues such as hair, nails and skin and feces, etc.), foods (cereals, vegetables, fruits, seafood, processed foods, etc.), beverages (water, tea, milk, fruit juice) , Alcoholic beverages, etc.), but is not particularly limited. Further, dilution and concentration are performed as needed.

[0010] The dye used in the present invention is preferably a dye which causes an association between the dye and the protein to be less likely to precipitate. Specifically, a dye having an ionic group that forms an ion associate having high solubility is preferable.

[0011] Dyes having an anionic group are particularly preferred, and can be electrically neutralized by binding to a positively charged site of a protein. For example, a dye having a hydrophilic group such as a sulfone group or a hydroxyl group may be used.

Further, in order to enhance the adsorptivity of the aggregate to the hydrophobic filter, the dye preferably has a hydrophobic group, and particularly preferably a dye having an aromatic ring.

Examples of such dyes include triphenylmethane dyes (eg, bromochlorophenol blue, tetrabromophenol blue, bromophenol blue, Coomassie brilliant blue, bromocresol purple), azo dyes (eg, buffalo black, Acid Orange 12, methyl orange, orange G, azosulfathiazole), xanthene dyes (eg, eosin, erythrosine) and the like.

Among them, bromochlorophenol blue (BCPB) and Coomassie brilliant blue (C
BB), tetrabromophenol blue (TBPB),
Bromocresol purple (BCP), bromophenol blue (BPB) and eosin are preferred.

[0015] The dyes surprisingly bind to almost all types of proteins and form ion associates.
Although the formation mechanism of the ion aggregate in the present invention is not clear,
It is considered to be either electrostatic attraction type ion association or hydrophobic structure type ion association [Honshu, "Comprehensive paper Design of separation and analysis method using ion association", Analytical Chemistry 38, 14
7-170, 1989].

In the present invention, this ion associate is completely dissolved in a solvent so as not to cause precipitation. Conditions can be easily set experimentally by those skilled in the art.

For example, Table 1 shows examples of conditions when an aqueous solution of human serum albumin (HSA) is used as the protein-containing solution.

[0018]

[Table 1]

When the sample and the dye are mixed, the dye may be mixed in the form of a solution containing the dye, or may be mixed in the form of a powder or a solid.

An ion associate is instantaneously formed by mixing the sample and the dye. The color tone of this ion associate is preferably different from that of the dye. For example, when BCPB, TBPB or BPB is used under the conditions of Table 1, the protein is yellow before the protein is added, but becomes blue after the protein is added.
Qualitative and quantitative analysis of proteins becomes possible by visual observation.

For example, as shown in FIG. 1, BCPB is -1 valent (HBCPB) at pH 4.1 or lower which is pKa _2.
^- ), Which shows yellow, but becomes -2 (BCPB ^2- ) by associating with the protein and shows blue.

As described above, it is particularly preferable that the change in the color tone is different from the absorption wavelength, such as from yellow to blue, because the change in the color tone can be easily visually recognized.

In such a case, it is preferable to add a buffer and set the pH in the solution to be lower than the pKa of the dye.

By passing the ion-associated solution through a hydrophobic filter, the ion-associated in the ion-associated solution is adsorbed on the hydrophobic filter.

Although the principle of the adsorption of the ion aggregate on the hydrophobic filter is not clear, it is considered that the electrically neutralized aggregate is hydrophobically adsorbed on the hydrophobic filter in the solution.

By selecting a dye having a small distribution ratio from the solvent to the hydrophobic filter, the unassociated dye is hardly adsorbed on the filter, so that the effect of the color of the unassociated dye is avoided. It becomes possible.

This eliminates the need for a washing operation for removing unassociated dye on the filter. For example,
In all combinations shown in Table 1, a washing step is not required (a nitrocellulose filter is used for the filter).

In the present invention, the amount of the ion aggregate adsorbed on the hydrophobic filter changes depending on the ratio of the amount of the sample to the amount of the dye to be mixed.

FIG. 2 shows this experimentally. The details of the operation of this experiment are described in Reference Example 2 later.

In FIG. 2, the horizontal axis represents the protein concentration in the mixed sample, the left vertical axis represents the difference in absorbance, and the right vertical axis represents the absorbance.

In FIG. 2, “○” indicates the absorbance of the ion association solution before passing through the hydrophobic filter at 602 nm specific to the ion association and the filtrate after passing through the hydrophobic filter. A value obtained by subtracting the absorbance (difference in absorbance), that is, the amount of the ion aggregate adsorbed on the hydrophobic filter.

The symbol “■” represents the absorbance of the ion association solution before passing through the hydrophobic filter at 602 nm specific to the ion association, that is, the amount of the ion association present in the ion association solution. Represent.

From this figure, it can be seen that the amount of ion associate formed in the ion associate solution obtained by mixing the sample and the dye increases as the protein concentration in the sample increases.

Further, the amount of the ion-associated substance adsorbed on the hydrophobic filter is determined when the protein concentration in the sample is about 30 mg / l.
Up to this point, it increases in accordance with the protein concentration in the sample, but at or above this concentration, it decreases.

Further, an experiment was conducted in which the coloring strength on the hydrophobic filter after passing the ion-associated solution through a hydrophobic filter and drying was measured with a densitometer. The result of this experiment is shown in FIG. The details of the experiment are described in Reference Example 3 below.

In FIG. 3, the horizontal axis represents the concentration of the protein in the sample mixed with the dye, and the vertical axis represents the level of absorbance at 602 nm (area of the peak of absorbance) specific to the ion associate.

From FIG. 3, it can be seen that, in this experimental system, the absorbance at 602 nm, which indicates the amount of ion association on the hydrophobic filter, is approximately 50% at the protein concentration in the sample.
It can be seen that the concentration increases up to mg / l in accordance with the protein concentration in the sample, but decreases above this concentration.

That is, in the present invention, when the amount of the protein is within a certain range relative to the amount of the dye at the time of forming the aggregate of the protein and the dye in the sample, the formed ion aggregate is applied to the hydrophobic filter. Although the protein is adsorbed, when the amount of protein increases beyond a certain range, it does not adsorb to the hydrophobic filter although the amount of ion aggregate formed increases.

In the present invention, examples of the material of the hydrophobic filter used include nitrocellulose, octadecylsilylated silica, octylsilylated silica, ethylsilylated silica, methylsilylated silica, phenylsilylated silica, fluorocarbonized silica, and graphite. And carbonized silica. Such a filter is commercially available, for example, a mixed cellulose ester filter A020A025A (nitrocellulose, pore size 0.1 mL).
2 μm, manufactured by Advantech Toyo).

The coloration on the filter may be measured visually or using a device such as a densitometer or a spot meter. The coloring intensity is determined, for example, by measuring the absorbance, transmittance, and fluorescence intensity in the visible region.

In the present invention, the color on the filter is different between when the hydrophobic filter is wet immediately after passing the ion association solution through the hydrophobic filter and when the hydrophobic filter is dry. different.

FIG. 4 shows this experimentally. The details of this experiment are described in Reference Example 4 below.

In FIG. 4, the horizontal axis represents the wavelength at the time of measuring the absorbance of the coloring on the hydrophobic filter, and the vertical axis represents the value of the absorbance at that time. The solid line in FIG. 4 shows the absorbance when the hydrophobic filter is wet immediately after passing the ion-associated solution through the hydrophobic filter, and the broken line shows the absorbance when the hydrophobic filter is dried.

From this figure, the coloring on the hydrophobic filter is
When dried than when hydrophobic filter is wet, HBCPB ^- the absorbance decreases near specific 434 nm (yellow), and the specific ion associate 602n
It can be seen that the absorbance in the vicinity of m (blue) has increased.

Therefore, it is preferable to measure the coloring on the filter after drying the filter. Drying of the filter may be performed by leaving, blowing, heating or the like. In the case of drying by leaving, it is usually sufficient to leave it for 20 minutes or more.

Reference Example 2, Reference Example 3, and Reference Example 4
From the results of (1), the following effects are presumed on the formation of the aggregate of the protein and the dye in the sample, the adsorption of the formed ion aggregate to the hydrophobic filter, and the coloring in the present invention.

The dye is monovalent (HB) at a pH of pKa ₂ or less.
CPB ⁻ ), but the dye becomes polyvalent (BCPB ²⁻ ) by associating with the protein. At this time, if the amount of the protein is at least a certain amount relative to the amount of the dye, the dye associated with the protein is multivalent (BCPB ²⁻ ) and associates with a specific limited site of the protein.

When the amount of the protein is less than a certain amount relative to the amount of the dye, that is, when the amount of the dye is excessive relative to the amount of the protein, the polyvalent dye (BCPB ²⁻ ) is associated with a specific site. The monovalent dye (HBCPB ⁻ ), which is present in excess, further non-selectively associates with the protein to form a multi-aggregate.

An ion associate with a polyvalent dye, which is formed when the amount of the protein is at least a certain amount relative to the amount of the dye, has a low affinity for the hydrophobic filter and is slightly adsorbed.

However, the multi-aggregate formed when the amount of the protein is less than a certain amount relative to the amount of the dye has a hydrophobic property because a monovalent dye is multiply associated with a polyvalent dye. And the adsorption rate to the hydrophobic filter is increased by the hydrophobic affinity.

As described above, although the amount of ion aggregates formed increases as the protein concentration in the sample increases, the amount of ion aggregates adsorbed on the hydrophobic filter decreases to a constant protein concentration. It is inferred from the fact that the situation changes from increasing to decreasing. (See FIGS. 2 and 3)

The multi-aggregate adsorbed on the hydrophobic filter has a polyvalent dye (BCPB ²⁻ ) [blue] associated therewith and a large number of monovalent dyes (HBCPB ⁻ ) [yellow] associated therewith. although indicate yellow-green because this changes the environment, such as pH or salt concentration by being placed in a dry state, monovalent dye (HBCPB ^-) cause proton dissociation or association to a particular site of the protein of The monovalent dye (HBCPB ⁻ ) is converted to a polyvalent dye (BCPB ²⁻ ) and changes to a multivalent dye color (blue).

[0053] The above means that, upon drying, a monovalent dye (HBCPB ^-) absorbance of the specific 434nm vicinity (yellow) is reduced, increasing absorbance near 602nm specific to ion associate (blue) Inferred from doing
(See FIG. 4)

As described in detail above, the method for analyzing proteins in a sample and the protein analysis kit of the present invention
By adjusting the amount of the dye and the like with respect to the amount of the protein in the sample to be analyzed, coloring can be caused on the hydrophobic filter only when the protein in the sample is in a specific concentration range. That is, it is possible to detect that the protein in the sample is in a specific concentration range.

Then, one sample is analyzed by using an analysis kit comprising a plurality of concentrations of dyes in which the concentration range in which coloring occurs is changed stepwise by adjusting the amount of the dyes and the like. Quantitative analysis of the protein in it can be performed.

The protein analysis kit of the present invention,
FIG. 5 shows an example of an analysis kit (analysis kit 1) for performing the method of the present invention. In the analysis, first, the hydrophobic filter 2 is fixed to the opening of the cylinder 3 without any gap. Next, the ion association solution in which the sample and the dye are mixed is injected into the cylinder 3 in advance, and the piston 5 is moved in the direction of the opening 4 (that is, the lever 6 is pushed) to thereby obtain the ion association solution. To the outside of the cylinder. At this time, the ion association contained in the ion association solution is adsorbed on the hydrophobic filter 2. Alternatively, the opening 4 is introduced into the ion association solution obtained by adding a dye to the sample, and the piston 5 is moved to the side opposite to the opening 4 (that is, the lever 6 is pulled), so that the ion association is formed inside the cylinder 3. A mode may be adopted in which the combined solution is sucked and the ion association is adsorbed on the hydrophobic filter 2.

Reference Example 1 (Determination of optimum pH) 7.45
× 10 ^-4 M bromochlorophenol blue (BCPB)
[Tokyo Kasei Co., Ltd.] 8ml aqueous solution and 2N NaCl aqueous solution 1
Then, distilled water was added thereto to make the total volume 500 ml. Next, the absorbance of the dye alone at 430 nm, the absorbance of the dye alone at 590 nm, and the absorbance at 600 nm when 0.0504 g of HSA was mixed to form an ion associate,
The measurement was performed while changing the pH. The result is shown in FIG. From these results, it is optimal that the pH is around 2.5 under these conditions.

[Reference Example 2] (Study of the amount of ion associate adsorbed on hydrophobic filter (1)) (1) Preparation of dye solution 2.034 mg of BCPB (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in distilled water, and 100 ml of the dye solution [35 μM BCPB
Solution] was prepared.

(2) Preparation of Buffer Solution 189 mg of monochloroacetic acid was dissolved in 90 ml of distilled water.
After adjusting the pH to 3.1 with hydrochloric acid, the total volume was adjusted to 100 ml to prepare a buffer [0.02 M monochloroacetic acid-hydrochloric acid buffer (pH 3.1)].

(3) Preparation of Sample HSA was prepared at 10, 20, 30, 40, 50, 100, 1
It was dissolved in physiological saline (0.9% aqueous sodium chloride solution) at a concentration of 50, 200, 250, 300, 350, and 400 mg / l to prepare 12 types of samples.

(4) Measurement 1 ml of the dye solution and 1 ml of the buffer were mixed, and 8 ml of each sample was mixed to prepare an ion-associated solution.

Next, the absorbance at 602 nm of each of these 12 types of ion association solutions was measured using U-2000.
Was measured with a spectrophotometer (manufactured by Hitachi, Ltd.).

Thereafter, 10 ml of each of these 12 kinds of ion association solutions was added to a nitrocellulose filter (pore size: 0.2 μm, diameter: 25 mm, product number: A020).
A025A, manufactured by Advantech Toyosha).

602 n of the 12 kinds of filtrates obtained here
The absorbance at m was measured in the same manner as above.

Then, in order to calculate the amount of the ion-associated substance adsorbed on the hydrophobic filter, a specific amount of the ion-associated substance was determined.
At 02 nm, the absorbance of the filtrate after passing through the hydrophobic filter was subtracted from the absorbance of the ion association solution before passing through the hydrophobic filter. The result is shown in FIG.

[Reference Example 3] (Study of the amount of ion associate adsorbed on the hydrophobic filter (2)) (1) Preparation of dye solution BCPB (manufactured by Tokyo Chemical Industry Co., Ltd.) (2.034 mg) was dissolved in distilled water and the total amount was dissolved. 100 ml of the dye solution [35 μM BCPB
Solution] was prepared.

(2) Preparation of Buffer Solution 189 mg of monochloroacetic acid was dissolved in 90 ml of distilled water.
After adjusting the pH to 3.0 with hydrochloric acid, the total volume was adjusted to 100 ml to prepare a buffer [0.02 M monochloroacetic acid-hydrochloric acid buffer (pH 3.0)].

(3) Preparation of Sample HSA was prepared in 5, 10, 15, 20, 25, 50, 75,
It was dissolved in physiological saline (0.9% aqueous sodium chloride solution) so as to be 100, 125, and 150 mg / l, and ten kinds of samples were prepared.

(4) Measurement 1 ml of the dye solution and 1 ml of the buffer were mixed, 1 ml of each sample was mixed with the mixture, and 2 ml of distilled water was further mixed to prepare an ion association solution.

Next, 5 ml of each of these 10 types of ion associated solution was sucked into a syringe, and a nitrocellulose filter (pore size: 0.2 μm) was placed in front of the syringe.
, Diameter: 25 mm, product number: A020A025A, manufactured by Advantech Toyo), and the ion-associated solution in the syringe was pushed by a piston and passed through a filter.

After drying these 10 types of filters in a dryer at 40 ° C. for 10 minutes, the colored 602 n
The absorbance height at m (area of the absorbance peak) is C
S-9300PC densitometer (manufactured by Shimadzu Corporation)
Was measured. The result is shown in FIG.

REFERENCE EXAMPLE 4 (Study of Coloring Spectrum Change on Hydrophobic Filter by Drying) (1) Preparation of Dye Solution BCPB (manufactured by Tokyo Chemical Industry Co., Ltd.) (2.034 mg) was dissolved in distilled water to make a total volume of 100 ml. Solution [35 μM BCPB
Solution] was prepared.

(2) Preparation of Buffer Solution 189 mg of monochloroacetic acid was dissolved in 90 ml of distilled water.
After adjusting the pH to 3.0 with hydrochloric acid, the total volume was adjusted to 100 ml to prepare a buffer [0.02 M monochloroacetic acid-hydrochloric acid buffer (pH 3.0)].

(3) Preparation of Samples HSA was adjusted to 50 mg / l with physiological saline (0.9%
(Aqueous sodium chloride solution) to prepare a sample.

(4) Measurement 1 ml of the dye solution and 1 ml of the buffer were mixed, 1 ml of the sample was mixed with the mixture, and 2 ml of distilled water was further mixed to prepare an ion association solution.

Next, 5 ml of this ion association solution was sucked into a syringe, and a nitrocellulose filter (pore size: 0.2 μm, diameter: 25 mm, product number: A020A025A, manufactured by Advantech Toyo) was attached to the tip of the syringe. ,
The ion association solution in the syringe was pushed by a piston and passed through a filter.

After drying this filter in a dryer at 40 ° C. for 10 minutes, the absorbance spectrum of the color on the filter
S-9300PC densitometer (manufactured by Shimadzu Corporation)
Was scanned and measured. The result is shown in FIG.

[0078]

According to the present invention, an extraction step, a coloring step,
Since the washing step is basically unnecessary, the number of steps is very small.The principle of ion association is adopted, so that color measurement can be performed in a short time. An excellent, simple and rapid means of analyzing proteins is provided.

In the analysis method and the analysis kit of the present invention, it is possible to detect that the protein in the sample is in a specific concentration range.

Further, by using an analysis kit comprising a plurality of dyes at different concentrations, quantitative analysis of proteins in a sample can be performed.

[0081]

[Example 1] (Analysis of protein in sample (1)) (1) Preparation of dye-containing solution BCPB (manufactured by Tokyo Chemical Industry Co., Ltd.) was adjusted to 372.5 μM with 1 M monochloroacetic acid-hydrochloric acid buffer. (PH 2.05) to prepare a dye-containing solution.

(2) Preparation of Sample HSA was prepared at 1.0, 2.5, 5.0, 10, 25, 50,
Dissolve in physiological saline (0.9% sodium chloride aqueous solution) so as to be 100, 200, 250, 500 mg / l,
Ten samples were prepared. A sample in which HSA was dissolved in urine so as to be 50 mg / l was also prepared.

(3) Measurement 0.5 ml of the dye-containing solution and 2 ml of each sample were mixed. Thereafter, 0.5 ml of each of these 11 kinds of mixed liquids was added to a nitrocellulose filter (pore size: 0.2
μm, manufactured by Advantech Toyo).

The color of this filter was measured visually.
If a blue spot was detected on the filter, it was determined that protein was present in the sample.

As a result, it was possible to determine visually up to a concentration of 2.5 mg / l. Further, a UV lamp was irradiated on the filter, and the fluorescence (red) was visually measured. As a result, it was possible to determine visually at all concentrations.

HSA in the urine sample was confirmed by both means of measuring the color of the filter and measuring the fluorescent color.

[Example 2] (Analysis of protein in sample (2)) (1) Preparation of dye solution 2.034 mg of BCPB (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in distilled water to make a total volume of 100 ml, and the dye solution [35 µM BCPB]
Solution] was prepared.

(2) Preparation of Buffer Solution 189 mg of monochloroacetic acid was dissolved in 90 ml of distilled water.
After adjusting the pH to 3.0 with hydrochloric acid, the total volume was adjusted to 100 ml to prepare a buffer [0.02 M monochloroacetic acid-hydrochloric acid buffer (pH 3.0)].

(3) Preparation of Sample HSA was prepared at 1.25, 3.75, 6.25, 10, 1
2.5, 16.25, 18.75, 22.5, 25, 3
Dissolve in physiological saline (0.9% aqueous sodium chloride solution) to a concentration of 7.5, 50, 75, or 100 mg / l.
Different types of samples were prepared. In addition, a sample composed of physiological saline was prepared as a control.

(4) Measurement 1 ml of the dye solution and 1 ml of the buffer were mixed, 1 ml of each of the samples was mixed, and 2 ml of distilled water was further mixed to prepare an ion association solution.

Next, 5 ml of each of these 14 kinds of ion association solutions was sucked into a syringe, and a nitrocellulose filter (pore size: 0.2 μm) was placed in front of the syringe.
, Diameter: 25 mm, product number: A020A025A, manufactured by Advantech Toyo), and the ion-associated solution in the syringe was pushed by a piston and passed through a filter.

These 14 types of filters were dried in a dryer at 40 ° C. for 10 minutes. The color of these filters was measured visually. The coloring on the filter is shown in FIG.

As a result, the protein concentration in the sample was 3.
Blue coloration was detected at 75 mg / l to 22.5 mg / l. That is, the presence of the protein in the sample of 3.75 mg / l to 22.5 mg / l was confirmed.

[Example 3] (Analysis of protein in sample (3)) (1) Preparation of dye solution 10.17 mg of BCPB (manufactured by Tokyo Chemical Industry Co., Ltd.);
53 mg, 15.98 mg, 17.43 mg, 20.3
4 mg, 21.79 mg, 23.24 mg, 29.05
mg or 40.67 mg dissolved in distilled water to give a total amount of 100 mg.
9 dye solutions [175 μM, 250 μM
, 275 μM, 300 μM, 350 μM, 375 μM,
400 μM, 500 μM, or 700 μM BCPB solution].

(2) Preparation of Buffer Solution 1.89 g of monochloroacetic acid was dissolved in 90 ml of distilled water.
After adjusting the pH to 3.1 with hydrochloric acid, the total volume was adjusted to 100 ml, and the buffer [0.2 M monochloroacetic acid-hydrochloric acid buffer (p
H3.1)] was prepared.

(3) Preparation of sample HSA was prepared in 10, 25, 50, 75, 100, 125,
It dissolved in physiological saline (0.9% sodium chloride aqueous solution) so that it might become 150, 175, 200, 300, and 400 mg / l, and 11 types of samples were prepared. In addition, a sample composed of physiological saline was prepared as a control.

(4) Measurement 1 ml of each of the nine dye solutions and 1 ml of the buffer
Were prepared as a set. 1 ml of each of the above 12 kinds of samples is mixed with each of the sets,
Further, 2 ml of distilled water was mixed to prepare a total of 108 kinds of ion associated solution.

Next, after sucking 5 ml of each of these 108 kinds of ion associated solution into a syringe, a nitrocellulose filter (pore size: 0.2 μm) was placed in front of the syringe.
m, diameter: 25 mm, product number: A020A025A, manufactured by Advantech Toyo), and the ion-associated solution in the syringe was pushed by a piston to pass through a filter.

These 108 types of filters were dried in a dryer at 40 ° C. for 10 minutes. The color of this filter was measured visually.

The coloring on these filters is shown in FIG. In FIG. 7, the horizontal axis represents the protein concentration in the sample, and the vertical axis represents the BCPB concentration in the dye solution. And
In FIG. 7, “●” indicates that the color on the hydrophobic filter is blue, “◎” indicates that the color on the hydrophobic filter is light blue, and “「 ”indicates that the hydrophobic filter is colored. It is not done.

FIG. 8 shows the results of measurement using a CS-9300PC densitometer (manufactured by Shimadzu Corporation).
The height of the absorbance (area of the peak of the absorbance) at 602 nm of the coloring on the 08 types of filters is shown.

From these results, in the present invention, by adjusting the amount of the dye with respect to the amount of the protein in the sample to be analyzed, only when the protein in the sample is in a specific concentration range, the hydrophobicity is increased. Coloring can occur on the filter. That is, it was confirmed that it was possible to detect that the protein in the sample was in a specific concentration range.

[Brief description of the drawings]

FIG. 1 is a diagram showing a relationship between pH and absorbance of a dye (430 nm, 590 nm) and an ion associate (600 nm).

FIG. 2 is a diagram showing the relationship between the protein concentration in a sample and the amount of ion aggregates and the like adsorbed on a hydrophobic filter.

FIG. 3 is a graph showing the relationship between the protein concentration in a sample and the amount of ion associate adsorbed on a hydrophobic filter.

FIG. 4 is a diagram showing coloring spectra when the hydrophobic filter is wet and when it is dry.

FIG. 5 is a protein analysis kit according to the present invention.

FIG. 6 is a diagram (photograph) showing coloring of a hydrophobic filter in a sample having each protein concentration.

FIG. 7: Protein concentration in sample and BC in dye solution
It is a figure showing coloring of a hydrophobic filter at the time of performing analysis by changing both PB concentrations.

FIG. 8: Protein concentration in sample and BC in dye solution
It is the figure which showed the height of the light absorbency of the coloring of the hydrophobic filter at the time of performing analysis by changing both PB concentrations.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Analysis kit 2 Hydrophobic filter 3 Cylinder 4 Opening 5 Piston 6 Lever

Claims (2)

[Claims] 1. a) mixing a sample which may contain a protein and a dye which forms an ion aggregate with the protein, and forming an ion aggregate solution without precipitating the ion aggregate; b A) a step of adsorbing the aggregate dissolved in the ion aggregate solution on the filter by passing the ion aggregate solution through a hydrophobic filter; and c) qualitatively and quantitatively determining the aggregate on the filter. A method for analyzing a protein in a sample, comprising sequentially performing the steps of: 2. A protein analysis kit for use in the method according to claim 1, comprising a dye that forms an ion aggregate with a protein; and a hydrophobic filter.