JPS6049256B2 - Shiyaku for protein analysis - Google Patents

Description

[Detailed description of the invention] The present invention relates to a reagent for protein analysis.

As a conventional reagent for protein analysis, a reagent described in Japanese Unexamined Patent Publication No. 53-2549 is known.

This reagent for protein analysis consists of a dye represented by the formula and PK
It consists of an acid of aO~4.

The reagent itself has a brownish color, but when the reagent and a sample containing protein are mixed, the dye represented by formula (1) binds to the protein and the mixture turns blue.
Then, the degree of coloration of the mixed solution is determined by absorbance, and the amount of protein contained in the sample is measured from that value. However, JP-A-53-2549
The protein analysis reagents described in Publication No. 0 include the following (1) to (
There are drawbacks shown in 3). (1) A mixture containing a protein-dye complex obtained by combining the dye represented by formula (1) with a protein has poor stability, and the degree of coloration of the mixture changes over time. The amount of protein measured differs depending on how many minutes after mixing the protein analysis reagent and sample, the absorbance is measured. This makes it difficult to accurately quantify the amount of protein contained in a sample.

(2) Depending on the degree of stirring or shaking when mixing the protein analysis reagent and the sample, the degree of coloration of the mixed solution will vary, and therefore the absorbance will also vary.

this is! It seems that this problem can be solved by always keeping the stirring or shaking conditions constant, but it is difficult to keep the stirring or shaking conditions constant because of the individual differences among the measurers. This makes it difficult to accurately quantify the amount of protein. 4(3) Protein analysis reagents are sensitive to a wide range of different proteins, but the degree of sensitivity (sensitivity)
There are variations in size, and for this reason, the slope of the calibration curve showing the relationship between absorbance and protein amount is not determined depending on the type of protein. Therefore, if a sample contains only one type of protein and the type of protein is known, the amount of protein contained in the sample can be accurately quantified using a calibration curve prepared in advance using that protein. However, if the sample contains two or more types of proteins, or if the sample contains one protein but the type of protein is unknown, use various proteins. The amount of protein in a sample to be determined differs depending on which of the various calibration curves created by the method is used as a standard, making it difficult to accurately quantify protein. As described above, the reagent for protein analysis described in Japanese Patent Application Laid-Open No. 53-2549 has various drawbacks, and it is currently desired to develop a reagent for protein analysis that does not have these drawbacks.

In view of the current situation, the present inventors have conducted extensive research in order to develop a reagent for protein analysis that does not have the above-mentioned disadvantages (1) to (3).

As a result, it was found that a desired reagent for protein analysis can be obtained when methylcellulose is blended into a mixture consisting of a dye represented by the above formula (1) and an acid. The present invention has been completed based on this knowledge. The reagent for protein analysis of the present invention retains the advantages of the reagent for protein analysis described in JP-A-53-254,
Moreover, the above-mentioned disadvantages (1) to (3) of the reagent are solved. That is, the reagent of the present invention is not easily influenced by substances coexisting in the sample, and can quantify various proteins contained in the sample with good reproducibility and excellent detection sensitivity. Furthermore, a mixed solution containing a protein-dye complex produced by mixing the reagent of the present invention and a sample is extremely stable. Therefore, even if 3 hours have passed since the reagent of the present invention and the sample were mixed, the amount of protein contained in the sample can be accurately quantified. Furthermore, by using the reagent of the present invention, it becomes possible to process large quantities of samples required for clinical testing. Further, even if the stirring or shaking conditions when mixing the reagent of the present invention and the sample change, the degree of coloration 1 of the mixed liquid is extremely difficult to change, and therefore the absorbance is also constant. Therefore, even if the stirring or shaking conditions used when mixing the reagent of the present invention and the sample are different, the amount of protein contained in the sample can be accurately measured. Furthermore, the reagent 5 of the present invention is sensitive to all of a wide range of different proteins, and its sensitivity to various proteins is also well constant, so that the slopes of the calibration curves for various proteins are extremely similar. Therefore, it goes without saying that there is only one type of protein contained in the sample and that protein is clearly 4, but there are also cases in which there are two or more types of proteins contained in the sample, or when there are two or more types of proteins contained in the sample. Even when the type of protein present in the sample is unknown, the total amount of protein in the sample can be measured with high accuracy. In addition, when measuring the absorption curve of the reagent of the present invention itself, the absorbance (blank value) at the protein measurement wavelength is low, which makes it possible to use a single-beam colorimeter even when the sample contains a large amount of protein. . The reagent of the present invention has various advantages as described above, and therefore, extremely good results can be obtained especially when quantifying a sample containing a low concentration of protein. In the reagent of the present invention, the active ingredient that changes the color tone is a known dye represented by the above formula (1), namely Coomassie Brilliant Blue (COOmassjeBril).
ljantB]Ue)G-250.

The amount of the dye of formula (1) to be contained in the reagent of the present invention is usually 0.004 to 0.02% (
The dye of formula (1) may be blended so that the amount (w/v), preferably 0.008 to 0.014%, is contained. The reagent of the present invention contains methylsenorelose.

The amount of methylcellulose contained in the reagent of the present invention is usually 0.01 to 0.5% (w/v), preferably 0.01 to 0.5% (w/v).
Methylcellulose may be blended in an amount of about 0.1%, particularly preferably about 0.01 to 0.03%. A wide range of known acids can be used as the acid to be mixed in the reagent of the present invention, and specifically, perchloric acid, hydrochloric acid, sulfuric acid, periodic acid,
Examples include inorganic acids such as phosphoric acid, phosphorous acid, selenic acid, and sulfurous acid, and organic acids such as oxalic acid, maleic acid, trichloroacetic acid, and dichloroacetic acid. Among the above acids, acids with a PKa of 0 to 4 are preferred, and acids with a PKa of 1 to 2 are particularly preferred. PKa
Among the acids having 0 to 4, periodic acid, phosphoric acid, phosphorous acid, selenic acid, sulfurous acid, maleic acid, oxalic acid, dichloroacetic acid, etc. are preferred, and phosphoric acid, oxalic acid, etc. are particularly preferred. The liquid nature of the reagent of the present invention should normally be acidic, preferably with a pH of 0.5 to 2. ＼Particularly preferably 0.7 to 0.9.
The amount of acid to be blended varies depending on the type of acid used, but cannot be generalized, but may be blended as appropriate to achieve the above pH. The dye of formula (1), methylcellulose and acid are added, for example, to an aqueous medium, preferably water.

There is no particular limitation on the order of addition of these components; for example, these components may be added to water at the same time, or these components may be added to water and then mixed.
An appropriate amount of a conventional buffer may be added to the reagent of the present invention in order to stabilize the pH of the reagent.

Further, an appropriate amount of a suitable solvent such as lower alcohols such as methanol and ethanol, dimethyl sulfoxide, dimethyl formamide, hexamethyl phosphoric triamide, etc. may be added to the reagent of the present invention. The reagent of the present invention exhibits excellent sensitivity to a wide range of different types of proteins.

Examples of such proteins include albumin (human serum albumin, bovine serum albumin), globulin (human serum γ-globulin, rabbit γ-globulin), hemoglobin (bovine 7 hemoglobin), chymotrypsinogen A1α-chymotrypsin, cytochrome C1α-amylase. , catalase, fibrinogen, histones, hemocyanin, pepsin, transferrin, trypsin, and the like. θ When quantifying the protein contained in a sample using the reagent of the present invention, the amount of the reagent of the present invention to be used varies depending on the amount of protein in the sample, but The reagent of the invention is preferably used in such a proportion that it is completely captured by the dye of formula (1) in the reagent.

For example, if there are 0 to 200 μg of protein in the sample to be quantified,
If contained, 0.004-0.02% (w/
It is preferable to use about 3 to 5 ml of the reagent of the present invention containing the dye of v). The reagent of the present invention is colored brown, but when the dye of formula (1) binds to protein, the mixture of the reagent of the present invention and the sample becomes blue.

A liquid mixture containing a protein-dye complex is extremely stable, and the degree of coloration is extremely difficult to change even when the liquid mixture is left for a long time of 3 hours or more. Furthermore, as the concentration of the protein-dye complex in the above mixture increases, the degree of blue coloration becomes stronger. Therefore, by measuring the degree of coloration of the mixture of the reagent of the present invention and the sample, the protein contained in the sample can be quantified. The method for measuring the degree of coloration of the liquid mixture is not particularly limited, and methods conventionally used in this field can be widely applied. For example, a spectrophotometer or 585-595n
The degree of coloration of the liquid mixture can be easily measured using an optical instrument such as a colorimeter that can measure the absorption of wavelengths in the m range.
More specifically, for example, the absorbance of the mixed solution can be measured using a spectrophotometer, and the amount of protein contained in the sample can be read from a calibration curve prepared in advance using a standard sample. The wavelength of the spectrum used during measurement is generally 585 to 595 nm, preferably 59 nm.
It is preferable to set it to about 0r1m. In the present invention, after mixing the reagent of the present invention and a sample, this mixed solution can be used as a sample for measurement with a spectrophotometer. The reagent of the present invention is suitably used for detecting proteins in urine, serum, cerebrospinal fluid, foods, other biologically derived fluids, and extracts thereof.

The present invention will be clearly explained with reference to Examples below.

In addition, the three bio-chemicals used for comparison in the examples
Rat ● Protein ● Assay ● Kit [trademark, manufactured by Bio Rat Laboratories Co., Ltd.] is published in Japanese Patent Publication No. 53-25.
The reagent is the same as the reagent described in the 49th official publication, and in the following examples, this commercially available product was diluted 5 times and then filtered (hereinafter referred to as 1BR kit diluted solution). Example 1 Oxalic acid is added to purified water, heated and stirred for 3 minutes, and then cooled to room temperature to prepare a 1.9 M/' oxalic acid aqueous solution.

The oxalic acid aqueous solution used below is the supernatant of such an oxalic acid aqueous solution.

Boil purified water, add methylcellulose [trademark Metrose 60SH501 manufactured by Shin-Etsu Chemical Co., Ltd.] so that the final concentration (concentration in the reagent for protein analysis of the present invention) is 0.025 W/■%, and stir once. Then add an equal amount of cooled purified water to prepare an aqueous methylcellulose solution.

Next, 20 ml of Coomassie Brilliant Blue G25O (trademark, manufactured by Sigma Corporation) is dissolved in the above 75 TrLL aqueous methylcellulose solution, and 75 ml of an oxalic acid aqueous solution is mixed with this solution.

The mixed solution is subjected to P-filtering twice, and the resulting Oki liquid is used as the reagent for protein analysis of the present invention. Example 2 3 ml of the reagent of Example 1 and 50 Pe of a standard sample (preliminarily mixed with human serum albumin at a ratio of 0.1 mg/ml to 1.0 m9/Mt) are mixed.

After 10 minutes, the absorbance was measured at a wavelength of 590 r1m using a spectrophotometer (Hitachi 12 Haru spectrophotometer, manufactured by Hitachi, Ltd.), and a calibration curve was created.

The resulting calibration curve is shown in FIG. Next, urine from a normal person or a patient with renal disease is collected, and the absorbance is measured in the same manner as above except that the urine 50Pe is used instead of the standard sample.

If necessary, dilute the urine to within the measurement range. The amount of protein in urine is determined from the measured absorbance and calibration curve. The results are shown in Table 1. Example 3 3 ml of the reagent of Example 1 and 50 μl of a sample containing human serum albumin at a ratio of 50 mg/De are mixed.

This mixing is done by hand shaking 5 times. The absorbance of the mixed solution 5 minutes to 3 hours after mixing is 59
Measure at 0 nm. FIG. 2 shows the relationship between time after mixing and absorbance. For comparison, 5 mL of BR kit diluted solution and 100 Pe of sample containing human serum albumin at a ratio of 50 m9/Df were mixed in the same manner as above, and the absorbance was adjusted to 5.
Measure at 95 nm.

FIG. 3 shows the relationship between time after mixing and absorbance. The following can be seen from Figures 2 and 3.

Coomassie Brilliant Blue G25O single serum albumin complex is immediately produced by mixing the reagent of the present invention and the sample. The mixed solution containing the complex is extremely stable even after 3 hours, and its absorbance remains constant. On the other hand, in the case of a mixture of the BR kit diluted solution and the sample, the absorbance remains almost constant for only about 5 to 3 minutes of the mixed solution, and after that, as time passes, the absorbance remains almost constant. The absorbance decreases considerably. The stability of the mixture of the reagent of the present invention and the sample is 6 times or more superior to that of the mixture of the BR kit diluent and the sample.

Example 4 A sample containing human serum albumin (concentration in the mixture of the sample and the reagent of Example 1 was 18 mg/dl)
human serum albumin prepared to give 50
Collect 1 sample and use 5 samples.

Add 3ml of the reagent of Example 1 to each of these 5 samples,
Shake by hand 5 times. After 5 minutes, the absorbance is measured at a wavelength of 59011 m.

The results obtained are shown in Table 2. Samples containing human serum albumin at different concentrations in the same manner as above (the concentration in the mixture of the sample and the reagent of Example 1 was 7.
Absorbance is measured in the same manner as above, except that human serum albumin (adjusted to 5 m9/dl) is used. The results are also shown in Table 2. In addition, samples containing human serum albumin (concentration in the mixed solution of sample and BR kit diluent are 18 mg/dl or 75 mg/dl)
Human serum albumin prepared to become De) 1
Absorbance is measured in the same manner as above except that 00Pe is collected and 5 ml of BR kit dilution solution is used.

The results are shown in Table 3. Y in Tables 2 and 3
is the average value of absorbance obtained by measuring the sample five times,
SD is the standard deviation, and CV is the variation determined by the following equation.

The following can be seen from Tables 2 and 3.

When the reagent of the present invention was used, the fluctuations (CV) at low and high concentrations were 1.3% and 0.6%, respectively, whereas when the BR kit diluted solution was used, The variation (CV) at low and high concentrations was 4.7% and 2.8%, respectively, and the variation when using the reagent of the present invention was extremely small compared to that when using the BR kit diluent. Therefore, the above results indicate that the protein content can be determined more accurately using the reagent of the present invention than when using the BR kit diluent. Example 5 A sample containing human serum albumin (concentration in the mixture of the sample and the reagent of Example 1 was 18 mg/De
human serum albumin prepared to give 50
Prepare 5 specimens by collecting 1 p' each.

3Tnt of the reagent of Example 1 was added to each of these five samples, and then vortexed
(VOltex-GENIEMOdelK-556G)
[trademark, manufactured by Scientific Industries, Inc.], stir for 5 seconds on the speed scale and leave to stand. After 5 minutes, 3 hours, and 3 hours after the mixture was left to stand, the mixture was stirred for 5 seconds at speed scale 2, and the absorbance was measured at a wavelength of 590 r1m after the mixture was left to stand for 5 minutes. The results obtained are shown in Table 4.
The absorbance is measured in the same manner as above except that stirring is performed at speed scale 5 instead of speed scale 2. The results obtained are shown in Table 5. **Absorbance is measured in the same manner as above except that stirring is performed at speed scale 8 instead of speed scale 2. The results obtained are shown in Table 6. In addition, 100 μ' of a sample containing human serum albumin (human serum albumin was adjusted so that the concentration in the mixture of the sample and BR kit diluent was 18 mg/d') and the BR kit diluent were collected. Stir at speed scale 2, 5, or 8 in the same manner as above except for using 5 ml, and measure the absorbance at a wavelength of 595 nm.

The results obtained under stirring conditions of speed scale 2 are shown in Table 7, and the results obtained under stirring conditions of speed scale 5 are shown in Table 8.
The results obtained under stirring conditions of speed scale 8 are shown in Table 9, respectively. Measurement conditions A, B, and C in Tables 4 to 9 are as follows.

A: Measure the absorbance 5 minutes after leaving it to stand B: Measure the absorbance 3 hours after leaving it C: After 3 hours have passed, stir again at the same speed scale for 5 seconds and then After standing for 5 minutes, the absorbance was measured.The following is clear from the results shown in Tables 4 to 9.

From each absorbance (measurement ratio data) under measurement conditions A, B, and C in Table 4, calculate SD, and from these values, calculate C.
When the V value is calculated, C■ (%)=3.3.

In addition, from each absorbance under measurement conditions A, B, and C in Tables 5, 6, 7, 8, and 9, X and SD
When the CV values are calculated, they are 5.1, 5.4, and 1, respectively.
2.5, 13.3, 13.6. In this way, the CV values of Yanghe using the reagent of the present invention (3.3, 5.1 and 5.4
) is the CV value (12.5) when using the BR kit diluent.
, 13.3 and 13.6), and this shows that the mixture of the reagent of the present invention and the sample is stable for a long time without being subject to changes over time. Next, Table 4 and 5
From the table and each absorbance under measurement conditions A, B, and C in Table 6 (45 pieces of data in total), Y=0.158. CD=0.
008 is found, and the CV value is calculated to be 4.9.

In contrast, measurement conditions A in Tables 7, 8, and 9,
From each absorbance in B and C (45 pieces of data in total), Y=
0.299, SD=0.039 was found, and the CV value was 13
．． It becomes 0. Therefore, when using the reagent of the present invention, it is possible to quantify the protein content in a sample more accurately than when using the BR kit diluent, and it is also possible to easily handle large quantities of samples that are currently required clinically. It can be processed quickly in assembly line work. Example 6 Standard samples containing various concentrations of human serum albumin, bovine serum albumin, bovine hemoglobin, or human serum γ-globulin are prepared.

50 Pe of a standard sample containing human serum albumin is added to 3 mL of the reagent of Example 1, shaken five times, and the absorbance is measured at a wavelength of 590 nm to create a calibration curve. Similarly, calibration curves are created for bovine serum albumin, bovine hemoglobin, and human serum γ-globulin. The calibration curve thus created is shown in FIG. Also, add human serum albumin to 5ml of BR kit diluent. Add 100 p' of a standard sample containing 100p' and shake 5 times, measure the absorbance at a wavelength of 595 nm and create a calibration curve.

Calibration curves are created in the same manner for bovine serum albumin and bovine hemoglobin. The calibration curve created in this way is
As shown in the figure. ; The curve connecting the / marks in Figures 4 and 5 is the calibration curve for human serum albumin, the curve connecting the A marks is the calibration curve for bovine serum albumin, and the curve connecting the Δ marks is the calibration curve for bovine serum albumin. This is the standard curve for hemoglobin, and the curve connecting the X marks is human serum 4
This is a calibration curve for γ-globulin. The following can be seen from FIGS. 4 and 3.

Both the reagent of the present invention and the diluted solution of the BR kit react with various proteins, but in the case of the reagent of the present invention, the reactivity with various proteins is almost the same, and therefore the slopes of the calibration curves of various proteins are extremely similar. are doing. Therefore, in cases where the sample contains only one type of protein and the type of protein is clear, there is no problem using either the reagent of the present invention or the BR kit diluent; If the protein species contained in the sample is unknown or if there are two or more protein species contained in the sample, the total protein contained in the sample can be extracted using the θBR kit diluent by using the reagent of the present invention. It can be said that quantification can be performed with higher accuracy than when using this method. Example 7 The absorbance of the reagent of Example 1 at a wavelength of 390 nm to 730 r1m is measured to create an absorption curve.

This absorption curve is shown in FIG. Further, the absorbance of the BR kit diluted solution at a wavelength of 410r1m to 700nm is measured to create an absorption curve. This absorption curve is shown in FIG. The following can be seen from FIGS. 6 and 7.

The absorbance (blank value) of the reagent itself of the present invention at the wavelength for protein quantification is about 1 compared to that of the BR kit diluent.
No.2. For this reason, when measuring absorbance using the BR kit dilution solution, there is no problem when using a double-beam spectrophotometer, but it should be especially noted when using a single-beam spectrophotometer. Accuracy deteriorates when the protein concentration contained in the sample is high. On the other hand, when using the reagent of the present invention, it is possible to quantify proteins in a sample with extremely high accuracy even when using a single beam spectrophotometer. Example 8 An appropriate amount of distilled water was heated to 70°C, and then methylcellulose [trademark Metrose 60SH50, manufactured by Shin-Etsu Chemical Co., Ltd.] 0
．． Add 4q and stir, then add Coomassie Brilliant.
0.24 q of Blue G25O (trademark, manufactured by Sigma), 27.4 ml of concentrated hydrochloric acid, and 7.38 V of anhydrous sodium acetate are added and stirred, and then distilled water is added to bring the total volume to 1 e.

This mixed solution is further stirred, left to stand for a day and night, and filtered, and the resulting liquid is used as the reagent for protein analysis of the present invention. The absorbance of the reagent obtained above at a wavelength of 370 r1m to 750 r1m is measured to create an absorption curve.

This absorption curve is shown in FIG. Comparative Example 1 A reagent for protein analysis is obtained in the same manner as in Example 8 above, except that 4 mL or 400 μe of Tween-20 (manufactured by Wako Pure Chemical Industries, Ltd.) is used instead of methylcellulose.

The absorbance of the obtained reagent at a wavelength of 370r1m to 750r1m is measured to create an absorption curve.

This absorption curve is shown in FIG. Comparative Example 2 NONIDET-P-40 instead of methylcellulose
[BITHESRARESERCH Lab. 4 m long
A reagent for protein analysis is obtained in the same manner as in Example 8 above, except that 1 or 400 μ' is used.

The absorbance of the obtained reagent at a wavelength of 370 nm to 750 r1m is measured to create an absorption curve.

This absorption curve is shown in FIG. Comparative Example 3 A reagent for protein analysis is obtained in the same manner as in Example 8, except that 4f1 or 0.4y of Brij35 (manufactured by Wako Pure Chemical Industries, Ltd.) is used instead of methylcellulose.

The absorbance of the obtained reagent at a wavelength of 370 nm to 750 nm is measured to create an absorption curve. This absorption curve is shown in FIG. Comparative Example 4 A reagent for protein analysis is obtained in the same manner as in Example 8, except that 4TrL1 or 400 μl of Triton XlOO (manufactured by Wako Pure Chemical Industries, Ltd.) is used instead of methylcellulose.

The absorbance of the obtained reagent at a wavelength of 370 nm to 750 nm is measured to create an absorption curve. This absorption curve is shown in FIG. The following can be seen from FIGS. 8 to 12.

The absorbance (blank value) of the reagent of the present invention itself at the wavelength for protein quantification (590 r1m) is low. On the other hand, in the protein analysis reagents of Comparative Examples 1 to 4, absorption was observed at 590 r1m, and the absorbance at 590 nm was significantly high. Therefore, when measuring protein in a sample using the protein analysis reagents of Comparative Examples 1 to 4, it is unavoidable that the accuracy deteriorates significantly, making it difficult to achieve the desired effect of the present invention. .

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the relationship between the amount of protein (human serum albumin) in a standard sample and absorbance measured using the reagent of the present invention.

Claims (1)

[Scope of Claims] 1. A reagent for protein analysis characterized by containing a dye represented by the formula ▲numerical formula, chemical formula, table, etc.▼, methylcellulose, and an acid as active ingredients. 2 The dye concentration is 0.004-0.02% (w/v), and the methylcellulose concentration is 0.01-0.5% (w/v).
/v). The reagent according to claim 1. 3 Dye concentration is 0.008-0.014% (w/v)
and the concentration of methylcellulose is 0.01-0.1%
(w/v) and the acid has a pKa of 0 to 4. 4. The reagent according to claim 2 or 3, which has a pH of 0.5 to 2.5.