JP2003262591A - Method for optimizing protein or nucleic acid quantification and method for quantifying substances by fluorescence measurement - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To optimize protein and nucleic acid quantification using an indicator and to quantify substances including fluorescence measurement so that proteins and nucleic acids can be accurately quantified visually. In addition, to provide a method for quantifying a substance that can perform more accurate quantification than before. SOLUTION: An operation of plotting, in a chromaticity coordinate system, a color generated by reacting a protein or the like with an indicator whose color changes by reacting with a protein or the like is performed by a plurality of parameters having different parameters affecting the generated color. Performed under conditions, the color generated at a desired concentration of protein or the like, including setting the parameters so as to be at or near the gray point in the chromaticity coordinate system, a method for optimizing measurement with an indicator such as a protein, In addition, in a method of quantifying a substance by fluorescence measurement, a method of quantifying a substance including expressing a measurement result in a chromaticity coordinate system and quantifying the substance based on a calibration curve on the chromaticity coordinate is provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for optimizing the quantification of proteins or nucleic acids and a method for quantifying substances by fluorescence measurement.

[0002]

2. Description of the Related Art Conventionally, indicators such as Coomassie Brilliant Blue (CBB) and Tetrabromophenol Blue (TBPB) have been used as a method for quantifying proteins in a trace amount. For example, CBB has an absorption maximum at 465 nm, but when bound to a protein, the absorption maximum shifts to 595 nm. The increase in absorbance at 595 nm at this time is proportional to the protein concentration. Therefore, the protein in the sample can be quantified by measuring the change in absorbance at 595 nm before and after adding the indicator to the sample.

A conventional protein quantification method using an indicator such as CBB is performed using a spectrophotometer. However, if the protein can be quantified with sufficient accuracy by visual observation, it is not necessary to use a spectrophotometer, which is very convenient and desirable. If proteins can be quantified visually without using a spectrophotometer, for example, for diagnostic agents, doctors and nurses can know the test results on the spot, and patients can also perform their own tests at home etc. It will be possible. Therefore, there is a demand for a method capable of quantifying a protein with high accuracy by visual observation.

On the other hand, conventionally, fluorescence resonance energy transfer
(Fluorescent Resonance Energy Transfer, FRET) is used to quantify substances. With FRET, the fluorescence energy of one excited fluorescent substance (donor) between two adjacent fluorescent substances is given to the other fluorescent substance (acceptor) as excitation energy, the fluorescence intensity of the donor decreases, and the acceptor becomes It is a reaction that emits light. It is used for three-dimensional structural analysis and quantitative analysis of protein complexes, and detection of nucleic acid hybridization.

However, in the general calibration curve by the conventional FRET method, the value obtained by dividing the fluorescence intensity of the donor by the fluorescence intensity of the acceptor is plotted on the vertical axis, but since this calibration curve is not generally a straight line, The accuracy of quantification by the FRET method is not always satisfactory.

[0006]

Therefore, an object of the first invention of the present application is to optimize the quantification of a protein or nucleic acid using an indicator so that the protein or nucleic acid can be quantified fairly accurately even by visual observation. Is to provide a way to do. Further, an object of a second invention of the present application is to provide a method for quantifying a substance including fluorescence measurement, such as the FRET method, which can perform a more accurate quantification than ever before. .

[0007]

As a result of earnest studies, the inventors of the present invention have found that in a method for quantifying a protein using an indicator, the color of which changes by reacting with a protein, a plurality of parameters that affect the resulting color are different. Under the conditions described above, plot the resulting color in the chromaticity coordinate system, and set the above parameters so that the color produced at the desired concentration of the test protein or nucleic acid is at or near the gray point in the chromaticity coordinate system. By doing so, it was found that the protein can be quantified fairly accurately by visual observation, and the first invention of the present application was completed.

That is, the first invention of the present application is to perform an operation of plotting a color generated by reacting a protein or nucleic acid with an indicator whose color changes by reacting with the protein or nucleic acid in a chromaticity coordinate system. , Under a plurality of conditions that have different parameters that affect the resulting color, and setting the parameters so that the color that occurs at the desired concentration of protein or nucleic acid is at or near the gray point in the chromaticity coordinate system. A method for optimizing the measurement of a protein or nucleic acid with an indicator is provided.

As a result of earnest research, the inventors of the present invention have found that
The inventors found that a calibration curve with good linearity can be obtained by plotting the fluorescence spectrum measured by the FRET method on the chromaticity coordinate system, and completed the second invention of the present application.

That is, the second invention of the present application relates to a method for quantifying a substance by fluorescence measurement, which comprises expressing the measurement result in a chromaticity coordinate system and quantifying based on a calibration curve on the chromaticity coordinate system. A quantification method is provided.

[0011]

As described above, the first invention of the present application is
It includes an operation of plotting the color generated by reacting a protein or nucleic acid with an indicator whose color changes by reacting with the protein or nucleic acid in a chromaticity coordinate system.
Here, the proteins and nucleic acids to be measured are not limited at all. For example, total proteins in body fluids such as urine, total nucleic acids in cell culture medium, etc. can be exemplified, but of course the present invention is not limited thereto. Also,
As the indicator for protein quantification, the above-mentioned CB
Examples thereof include, but are not limited to, B and TBPB, and 4-hydroxyazobenzene-2′-carboxylic acid and methyl orange. In addition, examples of indicators for quantifying nucleic acids include, but are not limited to, ethidium bromide, fluorescein, Cy Dye, SYBR Green and the like.

In the first invention of the present application, the color after the reaction of the indicator with the test protein or nucleic acid is plotted on the chromaticity coordinate system. Various chromaticity coordinate systems are known and are not particularly limited, but preferred chromaticity coordinate systems include a Q _x Q _y chromaticity coordinate system, an L * a * b * chromaticity coordinate system, and an xy chromaticity coordinate. The system can be mentioned.

Q _x Q _y chromaticity coordinates are chromaticity coordinates of complementary tristimulus values, and are represented by Reilly, CN et al., Anal. Chem., 32, 1218-
1232 (1960); Reilly, CN at al., Anal. Chem., 3
Coordinates proposed by 2, 1233-1240 (1960). Complementary color tristimulus values U, V, and W are calculated by the following equations.

[0014]

[Equation 1]

Here, A (λ) is the absorbance of the transmitting object at the wavelength λ, and P (λ) is the spectral distribution of the illumination light.

[Equation 2] Is a color matching function. Further, k in the formula is defined by the following formula.

[0016]

[Equation 3]

From the complementary color tristimulus values U, V and W, Q _x
And the value of Q _y can be calculated by the following formula.

[0018]

[Equation 4]

Further, the L * a * b * chromaticity coordinates are determined by the International Commission on Illumination.
The chromaticity coordinates defined by the (CIE) in 1976. The a * value and the b * value in the chromaticity coordinates can be calculated by the following formulas.

[0020]

[Equation 5]

In these equations, X _n , Y _n and Z _n respectively mean tristimulus values of the perfect diffusion surface, and other symbols are
It has the same meaning as the same sign in the case of Q _x Q _y chromaticity coordinates described above.

The xy chromaticity coordinates are chromaticity coordinates complementary to the above Q _x Q _y chromaticity coordinates and defined by CIE. The tristimulus value on the xy chromaticity coordinate can be calculated by the following formula.

[0023]

[Equation 6]

Here, T (λ) represents the transmittance at the wavelength λ, and other symbols have the same meanings as the same symbols in the above-mentioned Q _x Q _y chromaticity coordinates. The x value and the y value can be calculated from the above tristimulus values X, Y and Z by the following formulas.

[0025]

[Equation 7]

In the method of the first invention of the present application, the color after the reaction between the indicator and the sample is plotted in any of the chromaticity coordinate systems as described above. The color measurement and the plot on the chromaticity coordinate system can be performed by a known method, and a commercially available digital color analyzer (for example, Light Source Computer Images, Larkspur, California, USA,
This can be easily performed using a commercially available COLORTRON (trade name)) and the attached software and computer. In addition, the measurement value regarding color can be calculated from the obtained spectrum by using an ordinary spectrophotometer.

In the method of the first invention of the present application, the color is measured and plotted on the chromaticity coordinate under a plurality of conditions in which the parameters affecting the generated color are different. Here, examples of the parameter that influences the generated color include, but are not limited to, the concentration and pH of the indicator.

In the method of the first invention of the present application, at the desired concentration within the measurement range in which the test protein or the test nucleic acid in the sample is to be quantified, the above plot is displayed at or near the gray point of the chromaticity coordinate. Optimize the above parameters to fit. When the color is at the gray point, or when there is a color change that sandwiches the gray point, the color change can be recognized most sensitively with the naked eye, so that the color showing the quantitative result should be near the gray point. By doing so, it becomes possible to quantify with great accuracy even with the naked eye. Note that the gray point is a point where Q _x is about 0.31 and Q _y is about 0.32 in Q _x Q _y chromaticity coordinates, and _x is about _x in xy chromaticity coordinates.
0.31 and y are about 0.32. In these chromaticity coordinate systems, the "neighborhood" of the gray point differs depending on the required accuracy of the quantification, etc., but is preferably within a range of 0.04 radius from the gray point, more preferably within a radius of 0.02. is there. Also, in the L * a * b * chromaticity coordinate system, the gray point is the origin, and the “neighborhood” of the gray point
Is different depending on the required quantification accuracy and the like, but is preferably within a range of radius 5 from the gray point, and more preferably within a range of radius 3.

The "desired concentration" is the concentration that is most sensitively to be visually quantified. For example, in a test sample,
Concentration expected to be most frequent, most important concentration (for example, boundary between abnormal value and normal value in clinical samples)
Alternatively, the concentration is the center of the measurement range.

The operation for setting the parameters so that the color produced is near the gray point is performed by preparing a plurality of indicators having different parameters to be changed and measuring the test sample having a desired concentration using each indicator. , The points plotted on the chromaticity coordinates fall within or near the gray point.

Other conditions of the reaction, such as reaction temperature and reaction time, may be those conventionally used for the indicator. The reaction temperature is preferably room temperature, and the reaction time is not particularly limited, but is usually 30. Often about 2 seconds to 2 minutes.

In the second invention of the present application, in the method of quantifying a substance by fluorescence measurement, the measurement result is expressed in a chromaticity coordinate system and quantified based on a calibration curve on the chromaticity coordinate. As the chromaticity coordinates, various chromaticity coordinates as described above in the description of the first invention can be used, and xy chromaticity coordinates are particularly preferable.
The operation to plot the measurement result of the fluorescence measurement on the chromaticity coordinate is
Specifically, it can be performed as follows. Using a commercially available fluorescence altimeter, the fluorescence R was measured (the vertical axis is the fluorescence intensity, the horizontal axis is the wavelength of the light of 380-780 nm), and the reflectance R
(Intensity of scattered light with respect to incident light) is calculated. Using the calculated R value, the X, Y, and Z values are calculated by the following equation [Equation 7], and the x, y values are calculated by [Equation 8], and then plotted on the xy chromaticity coordinates.

[0033]

[Equation 8]

[0034]

[Equation 9]

The calibration curve can be prepared by performing fluorescence measurement on a plurality of standard samples of known concentrations and plotting the measurement results on the chromaticity coordinates as described above.

The fluorescence measurement in the second invention may be any method in which the substance is quantified by measuring fluorescence, such as a hybridized nucleic acid using the FRET method, fluorescence immunoassay, or fluorescence labeled probe. Can be exemplified, and a method for quantifying various substances such as metal ions using a fluorescent labeled probe can be exemplified, but the method is not limited thereto. Among these, in particular, in the FRET method, as specifically described in the following examples, it is possible to obtain an extremely linear calibration curve by plotting the measurement results on the xy chromaticity coordinates, and to obtain an accurate It is particularly preferable because it enables quantification. Incidentally, these fluorescence measuring methods themselves are well known and can be carried out by a conventional method. For example, the FRET method
The necessary reagents and equipment are commercially available and can be easily implemented.

According to the second invention of the present application, since a calibration curve having higher linearity than that of the conventional fluorescence measurement can be obtained, it becomes possible to perform the quantification with higher accuracy.

[0038]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Example 1 Optimization of protein quantification using CBB After dissolving 10 mg of CBB G-250 (manufactured by Nacalai Tesque, Inc.) in 5 mL of 95% ethanol, 10 mL of 85% phosphoric acid was added. Distilled water was added to this to make 100 mL (0.1 mM CBB G-250 8.5%
phosphoric acid). 200 μL of this CBB solution was dispensed into each well of the microtiter plate. 0, 0.1, 0.2, 0.3, 0.4
Alternatively, 0.5 μg / mL of human serum albumin (HSA, manufactured by Seikagaku Corporation, technical grade) solution was added to each well in an amount of 10 μL and mixed well. After 1 minute, a light absorption spectrum at 380 780 nm was measured with a microplate reader (Molecular device).

As shown in FIG. 1, the Q _x value at 0.3 mg / mL, which is the center of the measurement range, is about 0.37, the Q _y value is about 0.35, and the gray point (Q _x value is about 0.31, Q _y (The value is about 0.32)
It is slightly off from. Therefore, we optimized the color generated at 0.3 mg / mL to be near the gray point. The optimization was performed by changing the amount of phosphoric acid added to CBB to change pH, which is a parameter that affects the resulting color. The concentration of 0.3 mg / mL is the concentration on the boundary line between the normal value and the abnormal value of human urinary total protein, and this example is also a model for quantifying human urinary total protein.

The HSA concentration was fixed at 0.3 mg / mL, and the CBB concentration was
It was fixed at 0.1 mM and four different phosphate concentrations (8.5 14.5
%) Solution was prepared. The spectrum was measured by the same method as above, and the Q _x Q _y value was calculated.

The abscissa indicates the phosphoric acid concentration (%), and the ordinate indicates Q _x and
The Qy value is shown (Fig. 2). When the phosphoric acid concentration is 12%, Q _x , Q
Both y-values were closest to the gray point. The actual color development and the Q _x Q _y chromaticity coordinates under the set conditions are shown in FIG. In FIG. 3, the Q _x value at an HSA concentration of 0.3 mg / mL is about 0.30 and the Q _y value is about 0.32, which is close to the gray point. Digital Color Analyzer (COLORTRON
Since the measured color can be reproduced by using the software (product name)), the visual quantification can be easily performed by comparing the reproduced color with an actual color as a color sample. Further, since a linear relationship is established between the phosphoric acid concentration and the Q _x and Q _y values (FIGS. 2 and 4), even for concentrations other than 0.3 mg / mL, the accurate phosphoric acid concentration at the gray point can be known from these straight lines. FIG. 5 shows the relationship between this phosphoric acid concentration and HSA concentration. By using this curve formula, the gray point can be set freely between 0.1 and 0.5 mg / mL from the next time without performing an experiment.

Reference Example 1 Plot of protein quantification using TBPB on L * a * b * chromaticity coordinates TBPB is currently most frequently used in the visual protein quantification method, and yellow depending on the protein concentration, It changes to yellow-green, patina, and blue. However, the sensitivity is 1/1 of CBB G-250
It is about 0. TBPB (manufactured by Tokyo Chemical Industry Co., Ltd.) 24.64 mg to 1 mL of 95
After dissolving in 10% ethanol, 20 μL of 85% phosphoric acid was added. Distilled water was added to this to make 50 mL. This TB before use
The PB solution was further diluted 1/5 and used (0.1 mM TBPB-
0.0068% phosphoric acid). 200 μL of this 0.1 mM TBPB solution was dispensed into each well of a microtiter plate. 0 10mg /
10 μL of mL HSA solution was added to each well. 1 minute later, 380 with a microplate reader (Molecular device)
The absorption spectrum at 780 nm was measured. The L * a * b * value was calculated from the obtained spectrum by the following formula (FIG. 6).

Example 2 As an immunoassay sample by the FRET method, 0-100 μg / mL HSA (manufactured by Seikagaku Corporation,
100 μL of technical grade solution was added to each well.
Next, 7-amino-4-methylcoumarin-3-acetic acid (AMCA) labeled HSA (Jackson ImmunoResearch Laboratories, Inc.
10 μL) was added and mixed well. Next, fluorescein-labeled anti-HSA (BETHYL Laboratories, Inc.
40 μL) and well mixed, and then allowed to stand at room temperature for 10 minutes. 380-600nm fluorescence spectrum with excitation wavelength 350nm Microplate reader (Molecular device)
(Fig. 7). In the general calibration curve by FRET, the value obtained by dividing the fluorescence intensity of the donor by the fluorescence intensity of the acceptor is plotted on the vertical axis, but the calibration curve is not a straight line (Fig. 8).

Then, from the fluorescence spectrum according to the above formula,
The x and y values were calculated and plotted on the xy chromaticity coordinates. The results are shown in Fig. 9. As shown in FIG. 9, a calibration curve with extremely good linearity was obtained.

[0046]

The first aspect of the present invention provides a method for optimizing the quantification of a protein or nucleic acid using an indicator so that the protein or nucleic acid can be quantified fairly accurately even by visual observation. Therefore, the first invention of the present application enables easy quantification of proteins and nucleic acids.

Further, the second invention of the present application has provided a method for quantifying a substance including fluorescence measurement, such as the FRET method, which can perform a more accurate quantification than ever before. Therefore, according to the second invention of the present application, it has become possible to perform the fluorescence measurement more accurately than before.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of plotting the measurement results before optimization on the Q _x Q _y chromaticity coordinates in the protein quantification by CBB performed in Example 1.

FIG. 2 is a diagram showing the relationship between the phosphoric acid concentration and the Q _x value and the Q _y value when the phosphoric acid concentration added to the indicator is changed in the protein quantification by CBB performed in Example 1. .

FIG. 3 is a diagram showing the results of plotting the measurement results after optimization on the Q _x Q _y chromaticity coordinates in the protein quantification by CBB performed in Example 1.

FIG. 4 is a diagram showing the relationship between the phosphoric acid concentration and the Q _x value when the phosphoric acid concentration added to the indicator was changed in the protein quantification by CBB performed in Example 1. The four straight lines are from the bottom, HSA concentration 0.1, 0.
The results for 2, 0.4 and 0.5 mg / mL are shown.

[Fig. 5] Fig. 5 shows a protein (Q _{x =} 0.3) when Q _x was 0.3 in the protein quantification by CBB carried out in Example 1.
It is a figure which shows the relationship between A) concentration and phosphoric acid concentration.

FIG. 6 is a diagram showing a result of plotting protein quantification results using TBPB on L * a * b * chromaticity coordinates performed in Reference Example 1.

FIG. 7 is a diagram showing the result of immunoassay by FRET method (conventional method) performed in Example 2. The six curves show the HSA concentration from the bottom of the peak near the wavelength of 430 nm.
The results for 0, 10, 20, 50, 75 and 100 μg / mL are shown.

8 is a diagram showing a calibration curve (conventional method) for immunoassay by FRET method performed in Example 2. FIG.

FIG. 9 is a diagram showing a calibration curve in which the measurement results of the immunoassay by the FRET method performed in Example 2 are plotted on the xy chromaticity coordinates.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) G01N 33/50 G01N 33/50 P 33/53 33/53 D (72) Inventor Hiroaki Okabe Yokohama City, Kanagawa Prefecture 4-9-37 Tarumachi, Kohoku Ward Leopalace Tsunashima Daiichi 102 F term (reference) 2G020 AA08 DA02 DA03 DA04 DA05 DA13 DA16 DA23 DA31 2G043 AA01 BA16 DA02 EA01 FA06 FA07 GA25 GB28 KA02 KA05 NA11 2G045 AA40 BA11 DA13 DA36 FA11 FB12 JA01 2G054 AA10 CA22 CA23 EA03 GA04 4B063 QA01 QQ41 QR41 QR66 QS36 QX02

Claims (10)

[Claims] 1. An operation of plotting a color produced by reacting a protein or nucleic acid with an indicator whose color changes by reacting with the protein or nucleic acid in a chromaticity coordinate system, which affects the produced color An indicator for a protein or nucleic acid, comprising setting the parameters so that the color generated at a desired concentration of the protein or nucleic acid under a plurality of conditions with different parameters is at or near the gray point in the chromaticity coordinate system. Optimization method of measurement by. 2. The method of claim 1, wherein the chromaticity coordinate system is Q _x Q _y chromaticity coordinates. 3. The method of claim 1, wherein the chromaticity coordinate system is L * a * b * chromaticity coordinates. 4. The method of claim 1, wherein the chromaticity coordinate system is xy chromaticity coordinates. 5. The method according to any one of claims 1 to 4, wherein the parameter is the concentration and / or pH of the indicator. 6. An indicator optimized by the method according to any one of claims 1 to 5. 7. A method of quantifying a substance, which comprises quantifying a substance by fluorescence measurement, which comprises expressing the measurement result in a chromaticity coordinate system and quantifying based on a calibration curve on the chromaticity coordinate. 8. The method of claim 7, wherein the chromaticity coordinate system is an xy chromaticity coordinate system. 9. A method for quantifying a substance by fluorescence measurement,
The method according to claim 7 or 8, which is a quantification method using fluorescence resonance energy transfer. 10. The method according to claim 7, wherein the substance to be measured is a protein or a nucleic acid.