JP3090503B2 - Method for measuring protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring a protein. More specifically, the present invention relates to a method for measuring a protein in a sample such as urine, which is used in a field such as a clinical test.

[0002]

2. Description of the Related Art Conventionally, in the field of clinical examinations and the like, proteins in samples such as urine and cerebrospinal fluid have been frequently measured, and clinically important information has been obtained. For example, protein (albumin, globulin, etc.) in urine is found in normal individuals, but renal proteinuria in renal disease, cardiac proteinuria seen in heart failure, etc., cachexia appearing in leukemia, carcinoma, etc. Increased protein in urine during various diseases, such as proteinuria, febrile proteinuria seen in fever, cerebral hemorrhage, nervous proteinuria in epilepsy, and toxic proteinuria due to drug intoxication. It has been known. Normally, the urine excretion of protein in normal humans is 10 to 100 mg per day, and if it exceeds 150 mg per day, it is considered abnormal proteinuria, and clinical examination indicates 5 to 10 mg / dl.
Above this, it is determined that proteinuria is positive. Thus, the measurement (qualitative and quantitative) of protein in urine is an important test in clinical examination.

Hitherto, as a protein measuring method, the Kjeldahl method, the Lowry method, the buret method, the Coomassie brilliant blue method and the like have been known. In these methods, the Kjeldahl method is complicated in operation and unsuitable for daily routine work. The Lowry method is not suitable for measuring protein in urine because it is affected by amino acids in the sample and contains a large amount of amino acids in urine. The buret method has low sensitivity and cannot be applied to the measurement of trace components such as proteins in urine. Coomassie Brilliant
The blue method is unsuitable for continuous measurement because it is susceptible to contaminants in the sample, has a narrow measurable concentration range, and has a tendency for blue deposits to remain on the reaction tube and the measurement cell.

[0004]

In view of the problems of the conventional method, a complex comprising a metal and a dye (hereinafter referred to as a metal-dye complex for convenience) is used to determine the absorption wavelength of the complex in the presence of a protein. A method of measuring a protein using shift (hereinafter, referred to as a metal-dye complex method for convenience) has been proposed. For example, a complex composed of pyrogallol red and molybdate (maximum absorption: 470 nm) was shifted because the absorption wavelength of the complex was shifted to a longer wavelength side (maximum absorption: 604 nm) in the presence of a protein (such as albumin). It has been reported that a protein can be colorimetrically quantified by measuring absorbance at a wavelength (for example, analytical chemistry,
32, p. 379, 1983).

[0005] The metal-dye complex method has the advantages that a trace amount of protein can be measured with high sensitivity, there is no influence from contaminating amino acids, and there is little contamination of cells and the like. However, when this method is used to measure protein in urine and other samples, the measurement of protein is affected by the chelating components (eg, citric acid, oxalic acid, phosphoric acid, etc.) contained in the sample. Can not. That is, due to the chelating component in the sample, a part of the metal of the metal-dye complex is eliminated from the complex, forming a complex with the chelating component in the sample. Also showed a low value, indicating that the protein concentration was negative. For this reason, the method was excellent in measurement sensitivity but could not be put to practical use.

In order to avoid such a problem, a method of using a pyrogallol red-molybdenum complex in combination with a chelating agent and / or a metal capable of binding to a chelating component in a sample (usually referred to as a pyrogallol red method) is practical. Has been However, in the pyrogallol red method,
There is a problem that the reaction is slow and the measurement time is long (usually about 20 minutes). SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems of the prior art, and has as its object to provide a method for measuring a protein in a specimen such as a biological sample with high accuracy and speed.

[0007]

Means for Solving the Problems In view of the above circumstances, the present inventors have intensively studied a method for measuring a protein by a metal-dye complex method. As a result, the presence of a polyhydric alcohol at the time of measurement allows the chelating component to be eliminated. The present inventors have found that the protein can be measured with high accuracy and speed even in the case of the contained sample, and completed the present invention. That is, the measurement method of the present invention uses a complex comprising a metal and a dye, and uses a shift in the absorption wavelength of the complex in the presence of the protein to measure the protein (metal-dye complex method). It consists in performing the measurement in the presence of alcohol.

In the metal-dye complex used in the method of the present invention, the dye forms a complex with a metal to color (or increase the degree of coloring), and the presence of protein causes a shift in the absorption wavelength of the complex. Any dye can be used as long as it is obtained, and for example, pyrocatechol violet, pyrogallol red, galein and the like are preferably used. The amount of the dye used is usually 0.0005 to 0.1 (W / V)% of the dye concentration in the measurement sample solution [hereinafter, unless otherwise specified,% indicates (W / V)], preferably 0.001 to 0.1 (W / V). Adjusted to be 0.05%.

As the metal, various metals can be used as long as they form a complex with the above-mentioned dyes and are colored (or the degree of coloring is increased), for example, molybdenum, tin, iron and the like. Can be These metals are generally used in the form of oxyacid salts, halides, complex salts, organic or inorganic acid salts, and include, for example, molybdate (alkali metal salt, ammonium salt, etc.), stannate (alkali metal salt, Ammonium salt), tin chloride, tin sulfate, ferrate (alkali metal salt, ammonium salt, etc.), iron chloride and the like.
The amount of the metal used is usually adjusted so that the metal ion concentration in the measurement sample solution is 0.0005 to 0.1%, preferably 0.001 to 0.05%.

As the polyhydric alcohol used in the present invention, various polyhydric alcohols such as glycols and sugar alcohols can be used.
Sorbitol, dulcitol, glycerol, polyglycerol and the like. These polyhydric alcohols are 2
More than one species may be used in combination. The amount of polyhydric alcohol used is
It can be appropriately adjusted according to the content of the chelating component in the sample and the like, and is usually used by adjusting the concentration in the measurement sample solution to about 0.1 to 10%, preferably about 0.5 to 5%. . If the amount of the polyhydric alcohol used is less than 0.1%, the effect of addition is not recognized, and if it exceeds 10%, there is no problem. However, a sufficient effect can be obtained up to 10%. In the present invention, the polyhydric alcohol may be present at the time of measurement,
A polyhydric alcohol may be added to the reagent solution containing the metal-dye complex, or a polyhydric alcohol may be added to a sample such as urine. May be. Preferably, a polyhydric alcohol is added to the reagent solution containing the metal-dye complex in advance.

The method of the present invention can be carried out in the same manner as the conventional method except that the measurement is carried out in the presence of a polyhydric alcohol. As an example, first, a suitable solvent (e.g., glycine buffer or the like) is added to the dye and metal in a predetermined amount to form a metal-dye complex, and by adjusting the pH as necessary, A reagent solution containing the complex is prepared. This reagent solution is mixed with a protein-containing sample (diluted sample appropriately diluted as necessary) to prepare a measurement sample solution, which is kept at about room temperature for a certain period of time (for example, about 1 to 10 minutes).
put. Since the absorption wavelength of the metal-dye complex shifts depending on the protein, the absorbance at the shifted wavelength is measured. On the other hand, the same operation is performed using a standard sample solution having a known protein concentration instead of the sample, and the absorbance at the shifted wavelength is measured to prepare a calibration curve. By comparing this calibration curve with the absorbance obtained with the above-mentioned sample, the protein concentration in the sample can be determined. The present invention is not limited to the above method, but can be applied to any of the metal-dye complex methods.

In the present invention, it is preferable to use a surfactant (eg, sodium lauryl sulfate, Triton X-405, etc.) in order to prevent contamination of the reaction tube, measurement cell, and the like. The surfactant is preferably added in advance to the reagent solution containing the metal-dye complex, and the amount of the surfactant to be used is adjusted so that the surfactant concentration in the reagent solution is about 0.1 to 2%. Good.

The measuring method of the present invention is used for measuring a protein in a sample such as urine or cerebrospinal fluid, and particularly for a short time (usually 1 to 10
(Approximately minutes), which is suitable for measurement using an automatic analyzer. Further, the present invention can be applied to the measurement of protein by a test paper method.

[0014]

EXAMPLES Hereinafter, the present invention will be described in more detail based on Examples.
Explain, but the invention is not limited to these examples
Absent. Example 1Creating a calibration curve  Mix the following reagents in purified water and adjust the pH to 2.2 with hydrochloric acid
This was adjusted to prepare a reagent solution having the following composition. Reagent: glycine 100 mM ammonium molybdate 0.006% pyrocatechol violet 0.005% mannitol 1.0% Triton X-405 1.0% Using human serum albumin as a protein standard solution, 300 mM
It was prepared to be g / dl. Using this standard solution, 30, 60,
Dilution series of 90, 150, 240, 300 mg / dl were prepared. This inspection
Add 200 μl of reagent solution to 5 μl of the body, and add
The absorbance at 75 nm was measured. Use purified water instead of the above sample
Perform the same operation with the albumin concentration of 0 mg / dl.
Absorbance was determined. Absorbance for each albumin concentration
Plotted to create a calibration curve. Fig. 1 shows the results.
You. As is clear from FIG. 1, over a wide concentration range
A good calibration curve having linearity was obtained.

Embodiment 2Measurement of protein in human urine  In the same operation as in Example 1, for 10 human urine samples,
The protein concentration was measured. The results are shown in Table 1. table
As shown in FIG. 1, the method of the present invention exhibits a negative value.
And good measurement values were obtained. As a comparative example,
The protein concentration of the same sample was determined using the conventional pyrogallol red method.
(Using a commercially available reagent). Table 1 shows the results.
Also shown. The correlation coefficient between the method of the present invention and the conventional method is 0.96
5 (regression equation Y = 0.951X-0.005), indicating good correlation
did.

Table 1

[0017]

As described above, in the method for measuring the protein of the present invention, the measurement value does not show a negative value because it is not affected by the chelating component in the sample. Also,
Measurement can be performed in a short time. Therefore, according to the method of the present invention, there is an effect that even a small amount of protein in a sample can be measured with high accuracy and speed.

[Brief description of the drawings]

FIG. 1 is a diagram showing a calibration curve according to the measurement method of the present invention.

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/68

Claims (5)

(57) [Claims] 1. Use of a complex comprising a metal and a dye,
A method for measuring a protein using a shift of the absorption wavelength of the complex in the presence of the protein, wherein the measurement is performed in the presence of a polyhydric alcohol. 2. The polyhydric alcohol is mannitol,
2. The method for measuring a protein according to claim 1, wherein one or more selected from sorbitol, dulcitol, glycerol and polyglycerol are used. 3. The method according to claim 1, wherein the pigment is pyrocatechol violet or pyrogallol red.
A method for measuring protein . 4. The method for measuring a protein according to claim 1, wherein the metal is molybdenum, tin or iron. 5. A protein comprising a complex comprising a metal and a dye,
Using the shift of the absorption wavelength of the complex in the presence of white matter
Reagent used in a method for measuring protein,
Characterized in that the reagent contains a polyhydric alcohol
Reagent for protein measurement.