WO2005024430A1 - タンパク質の分析方法およびそれに用いるタンパク質分析試薬 - Google Patents
タンパク質の分析方法およびそれに用いるタンパク質分析試薬 Download PDFInfo
- Publication number
- WO2005024430A1 WO2005024430A1 PCT/JP2004/012481 JP2004012481W WO2005024430A1 WO 2005024430 A1 WO2005024430 A1 WO 2005024430A1 JP 2004012481 W JP2004012481 W JP 2004012481W WO 2005024430 A1 WO2005024430 A1 WO 2005024430A1
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- WO
- WIPO (PCT)
- Prior art keywords
- ion
- chaotropic
- sample
- protein
- analysis
- Prior art date
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6827—Total protein determination, e.g. albumin in urine
- G01N33/683—Total protein determination, e.g. albumin in urine involving metal ions
- G01N33/6833—Copper, e.g. Folin-, Lowry-, biuret methods
Definitions
- the present invention relates to a protein analysis method and a protein analysis reagent used for the method.
- the Biuret method is a protein analysis method utilizing the fact that a protein reacts with copper ions to form a complex and thereby develop a purple color.
- concentration of the protein can be measured by optically measuring the color reaction as absorbance or reflectance (for example, see Non-Patent Document 1).
- O Copper sulfate is used as the copper ion source.
- Non-patent document 1 "Chemistry for Experimental Chemistry, Protein I Separation and Purification'Properties" The Japan Biochemical Society, Tokyo Chemical Co., Ltd. 1990 p. 91
- the Biuret method has a problem that the viscosity of the reaction solution increases due to aggregation of proteins. This problem of increased viscosity is a serious problem for high-concentration protein samples.
- a sample analysis device hereinafter also referred to as a test strip
- a biological sample such as urine or blood is supplied as it is. Because of the reaction, the protein concentration in the reaction solution is high. Therefore, if the color of the Biuret reaction is analyzed optically in a highly viscous state, a problem arises in the accuracy of the analysis. [0005] Therefore, an object of the present invention is to provide a method for analyzing a protein having excellent analysis accuracy.
- a method for analyzing a protein of the present invention is a method for analyzing a protein in a sample, wherein the sample is alkaline and contains salts containing chaotropic ions and divalent salts.
- Biuret reaction is caused by adding copper salts, and this Biuret reaction is analyzed optically.
- the protein analysis method of the present invention is excellent in analysis accuracy because protein aggregation is prevented and the resulting increase in viscosity is prevented. According to the present invention, in particular, even if the protein sample has a high concentration, the viscosity of the reaction solution can be prevented from increasing, and the protein concentration can be analyzed with high accuracy.
- FIG. 1 is a graph showing a correlation between Example 1 and Comparative Example 1.
- the present inventors have repeated a series of studies, mainly on elucidation of the cause of protein aggregation, in order to achieve the above object.
- copper sulfate which is conventionally used in the Biuret method, is the cause of aggregation of proteins.
- salts containing strong pick ions prevents protein aggregation, and the present invention has been achieved.
- chaotropic ion divalent copper salts as the salts containing chaotropic ions and the divalent copper salts.
- examples of the chaotropic ion include SCN ion, I ion, NO ion, CIO ion, Br ion, C1 ion and the like. Preferred in this
- the divalent copper salts include CuSO, Cu (C10), Cu
- the divalent copper salts of the chaotropic ion include, for example, Cu (NO 2), Cu (C 10), CuBr and CuCl. Of these, the preferred
- the amount of the chaotropic ion to be added is, for example, in the range of 0.5 mM to 100 mM, and preferably in the range of ImM to 10 mM.
- the amount of the divalent copper salt added is, for example, in the range of 0.5 mM to 100 mM, preferably ImM It is in the range of 10 mM.
- the amount of the divalent copper salt of the chaotropic ion added is, for example, in the range of 0.5 mM to 100 mM. , Preferably in the range of ImM-lOmM.
- the dilution concentration of the sample is, for example, 1Z50 dilution.
- the sample may be used without dilution in the liquid analysis, or the sample may be diluted and used in the dry analysis.
- the liquid analysis is an analysis technique using a reagent solution, and is used, for example, in general experiments and the like.
- the dry analysis is an analysis method using a reagent or the like in a dry state, for example, an analysis method applied to a sample analysis tool (or a test piece) used in a clinical test or the like.
- the amount of the chaotropic ion to be added is, for example, 100 mM to 2000 mM. Range, preferably in the range of 500 mM to 100 mM.
- the amount of the divalent copper salt added is, for example, in the range of lOOmM to lOOOOmM, and is preferably Is in the range of 200mM-500mM.
- the amount of the divalent copper salt of the chaotropic ion added is, for example, in the range of 100 mM-1000 mM, and is preferably Is in the range of 200mM-500mM.
- alkaline agent examples include NaOH, KOH, LiOH, Ca (OH) and the like. Any of these alkaline agent
- a particularly useful force for processing into analytical tools is LiOH.
- the sample is alkaline at the time of the reaction between the chaotropic ion and divalent copper ion and the protein in the sample.
- the alkaline agent as described above may be mixed at the time of mixing the chaotropic ion and the divalent copper ion with the sample.
- the pH of the alkaline sample during the reaction is, for example, in the range of pH 9 or higher, preferably in the range of pH 11 or higher.
- the protein concentration in the sample is, for example, in the range of 0 to 15 g / dL (100 mL). This range is equivalent to the concentration range for protein measurement in the normal Biuret reaction.
- protein is intended to include a polypeptide.
- the optical analysis method includes, for example, analysis of the degree of coloring of the sample by a Biuret reaction.
- the analysis of the degree of coloring of the sample may be a visual determination, or may be an absorbance measurement or a reflectance measurement by an apparatus.
- the optical analysis method may be either qualitative analysis or quantitative analysis.
- the reagent of the present invention is a protein analysis reagent used in the protein analysis method of the present invention, wherein the reagent contains a chaotropic ion-containing salt and a divalent copper salt. It is.
- the chaotropic ions and the divalent copper salts include those described above.
- the reagent may contain other reagents such as an alkaline agent in addition to salts containing chaotropic ions and divalent copper salts. Examples of other reagents such as the alkaline agent include those described above.
- the reagent of the present invention is a protein analysis reagent used in the protein analysis method of the present invention, and is a reagent containing a divalent copper salt of a chaotropic ion.
- examples of the divalent copper salts of the chaotropic ion include those described above.
- the reagent may contain other reagents such as an alkaline agent in addition to the divalent copper salts of chaotropic ions.
- examples of other reagents such as the alkaline agent include those described above.
- the reagent of the present invention can be used, for example, by dripping it onto a sample, freeze-drying it on a microplate to dry it, and placing it on a sample analysis tool as described later.
- the sample analysis device of the present invention is used for the protein analysis method of the present invention, and includes a carrier and a reagent, and the reagent includes salts containing chaotropic ions and divalent copper salts. It is a sample analysis tool including.
- the chaotropic ions and the divalent copper salts include those described above.
- the reagent may contain other reagents such as an alkaline agent, in addition to salts containing chaotropic ions and copper (II) salts.
- Other reagents such as the alkaline agent include those described above.
- the sample analysis device of the present invention is used for the protein analysis method of the present invention, and includes a carrier and a reagent, and the reagent includes a divalent copper salt of a chaotropic ion. It is a tool.
- the divalent copper salts of chaotropic ions include those described above.
- the reagent may include another reagent such as an alkaline agent in addition to the divalent copper salts of chaotropic ions.
- Previous Other reagents such as the alkaline agent include those described above.
- the method for analyzing a protein according to the present invention can be carried out, for example, as follows.
- a sample containing a protein such as blood is suspended in water if necessary, and an alkaline agent is added to adjust the pH to the above-mentioned predetermined range.
- an alkaline agent is added to the sample solution and stirred, the protein and copper ion in the sample form a complex, and after the addition of the reagent, for example, 10 seconds. Color develops in 15 minutes. This change may be confirmed visually or may be measured using an optical system measuring device such as a spectrophotometer. By measuring the degree of this color development, qualitative or quantitative analysis of the protein is possible.
- the protein concentration can be determined from the degree of coloring of the Biuret reaction.
- the reaction temperature ranges, for example, from room temperature to about 37 ° C., which is a normal adjustment temperature in a general analyzer.
- a reagent solution is prepared by dissolving a divalent copper salt of a picky ion in alkaline water. The pH of this reagent solution is adjusted to the above-mentioned range when the sample is added. Then, a porous carrier such as a filter paper is impregnated with the reagent solution, and then dried.
- the carrier is not particularly limited. For example, a plastic porous sheet may be used in addition to the filter paper.
- the carrier supporting the reagent as described above may be used as a sample analysis tool as it is, or the carrier may be further supported on another substrate sheet or the like to be used as a sample analysis tool.
- the color develops, for example, for 10 seconds and 15 minutes after the sample is supplied.
- Qualitative or quantitative analysis of the protein is possible by visually confirming the degree of the color development or by measuring with a reflectometer. For example, if a calibration curve is prepared in advance, the concentration of protein can be determined by the coloring degree of the Biuret reaction.
- the carrier may be, for example, a substance having a flow path of a sample solution formed of a light-transmitting substance.
- a reagent containing a divalent copper salt of a chaotropic ion in the flow path of the solution You should place ⁇ .
- microplate reagents were prepared, and 50 ⁇ L each was dispensed into a 96-well mic opening plate and lyophilized.
- Example 2 5 ⁇ L of the same sample as in Example 1 and 350 ⁇ L of the following solution-based reagent were mixed and reacted at room temperature for 10 minutes, and then the absorbance at a wavelength of 546 nm was measured.
- an automatic analyzer (trade name: 7070, manufactured by Hitachi, Ltd.) was used.
- FIG. 1 is a graph showing the results of correlation between Example 1 and Comparative Example 1, and FIG. The equation shows an equation representing the correlation, and R 2 shows a correlation coefficient.
- Example 1 is a dry method
- Comparative Example 1 is a solution method.
- Example 1 As shown in FIG. 1, a high correlation was observed between Example 1 and Comparative Example 1.
- HSA human serum albumin
- the following reagent solution A was prepared, and 200 ⁇ L of the reagent solution A and 400 ⁇ L of the sample were reacted at room temperature for 8 minutes. Then, the reaction solution was dispensed into a transparent cell having a cell length of lmm, and the absorbance at a wavelength of 550 nm was measured with a spectrophotometer (trade name: V-550, manufactured by JASCO Corporation). The results are shown in Table 1 below.
- Lithium hydroxide 1.89g (10. OOOM)
- Lithium hydroxide 1.89g (10. OOOM)
- a force that could not be dispensed was a force that could not be moved and a force that could not aspirate the reaction solution with a pipette.
- the protein analysis method of the present invention is excellent in analysis accuracy because protein aggregation is prevented and the resulting increase in viscosity is prevented. Therefore, according to the analysis method of the present invention, it is possible to improve the accuracy of protein analysis in all fields such as medicine, pharmacy, and biochemistry, and in particular, fields where high-concentration proteins need to be analyzed, for example, It is useful for protein analysis using a sample analysis device in a clinical test.
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Food Science & Technology (AREA)
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- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2005513631A JPWO2005024430A1 (ja) | 2003-09-03 | 2004-08-30 | タンパク質の分析方法およびそれに用いるタンパク質分析試薬 |
US10/547,102 US20060177938A1 (en) | 2003-09-03 | 2004-08-30 | Method for analyzing protein and protein analysis reagent to be used therein |
EP04772437A EP1662260A4 (en) | 2003-09-03 | 2004-08-30 | PROTEIN ANALYSIS METHOD AND PROTEIN REAGENT USED IN SUCH A METHOD |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2003-311624 | 2003-09-03 | ||
JP2003311624 | 2003-09-03 |
Publications (1)
Publication Number | Publication Date |
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WO2005024430A1 true WO2005024430A1 (ja) | 2005-03-17 |
Family
ID=34269706
Family Applications (1)
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PCT/JP2004/012481 WO2005024430A1 (ja) | 2003-09-03 | 2004-08-30 | タンパク質の分析方法およびそれに用いるタンパク質分析試薬 |
Country Status (5)
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US (1) | US20060177938A1 (ja) |
EP (1) | EP1662260A4 (ja) |
JP (1) | JPWO2005024430A1 (ja) |
CN (1) | CN1777811A (ja) |
WO (1) | WO2005024430A1 (ja) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102944525B (zh) * | 2012-11-02 | 2015-03-11 | 上海市东方医院 | 镍-双缩脲试剂盒的制备方法、产品及应用方法 |
CN106885778B (zh) * | 2017-03-24 | 2019-12-27 | 依科赛生物科技(太仓)有限公司 | 一种提高蛋白浓度测定准确性的快速测定试剂及其测定方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6418059A (en) * | 1987-06-22 | 1989-01-20 | Eastman Kodak Co | Analysis method and element for protein analysis |
JPH1019898A (ja) * | 1996-07-01 | 1998-01-23 | Internatl Reagents Corp | 総蛋白質の定量方法および定量用試薬 |
JP2001099826A (ja) * | 2000-08-25 | 2001-04-13 | Internatl Reagents Corp | 錯体の発色方法および発色用試薬 |
JP2002350448A (ja) * | 2001-05-30 | 2002-12-04 | Kanto Chem Co Inc | 総蛋白質の定量方法及び定量用試薬 |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3310382A (en) * | 1963-02-04 | 1967-03-21 | George R Kingsley | Biuret reagent |
US3807956A (en) * | 1972-08-30 | 1974-04-30 | First Nat Bank Of Miami | Modified biuret reagent for glass storage and method |
US4132528A (en) * | 1978-01-03 | 1979-01-02 | Eastman Kodak Company | Analytical element for the analysis of liquids under high pH conditions |
US4337064A (en) * | 1979-12-21 | 1982-06-29 | Sherwood Medical Industries Inc. | Albumin determination method |
EP0054056A1 (en) * | 1980-06-23 | 1982-06-23 | Beckman Instruments, Inc. | Instantaneous rate kinetic method for determining total protein in cerebrospinal fluid |
JPS5885163A (ja) * | 1981-11-17 | 1983-05-21 | Fuji Photo Film Co Ltd | 蛋白質分析用一体型多層分析要素 |
JPS6252467A (ja) * | 1985-08-31 | 1987-03-07 | Konishiroku Photo Ind Co Ltd | タンパク質分析用多層分析素子 |
-
2004
- 2004-08-30 CN CN200480010702.2A patent/CN1777811A/zh active Pending
- 2004-08-30 JP JP2005513631A patent/JPWO2005024430A1/ja active Pending
- 2004-08-30 EP EP04772437A patent/EP1662260A4/en not_active Withdrawn
- 2004-08-30 US US10/547,102 patent/US20060177938A1/en not_active Abandoned
- 2004-08-30 WO PCT/JP2004/012481 patent/WO2005024430A1/ja active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6418059A (en) * | 1987-06-22 | 1989-01-20 | Eastman Kodak Co | Analysis method and element for protein analysis |
JPH1019898A (ja) * | 1996-07-01 | 1998-01-23 | Internatl Reagents Corp | 総蛋白質の定量方法および定量用試薬 |
JP2001099826A (ja) * | 2000-08-25 | 2001-04-13 | Internatl Reagents Corp | 錯体の発色方法および発色用試薬 |
JP2002350448A (ja) * | 2001-05-30 | 2002-12-04 | Kanto Chem Co Inc | 総蛋白質の定量方法及び定量用試薬 |
Also Published As
Publication number | Publication date |
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CN1777811A (zh) | 2006-05-24 |
EP1662260A4 (en) | 2006-10-25 |
JPWO2005024430A1 (ja) | 2006-11-02 |
EP1662260A1 (en) | 2006-05-31 |
US20060177938A1 (en) | 2006-08-10 |
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