WO2011071155A1 - 塩基性ペプチドの検出方法および塩基性ペプチド検出用試薬 - Google Patents

塩基性ペプチドの検出方法および塩基性ペプチド検出用試薬 Download PDF

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WO2011071155A1
WO2011071155A1 PCT/JP2010/072256 JP2010072256W WO2011071155A1 WO 2011071155 A1 WO2011071155 A1 WO 2011071155A1 JP 2010072256 W JP2010072256 W JP 2010072256W WO 2011071155 A1 WO2011071155 A1 WO 2011071155A1
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peptide
basic peptide
denatured
solution
albumin
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PCT/JP2010/072256
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English (en)
French (fr)
Japanese (ja)
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博幸 加畑
高橋　英樹
丸山　征郎
礼奈 鶴岡
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シスメックス株式会社
国立大学法人鹿児島大学
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Priority to JP2011545267A priority Critical patent/JP5632391B2/ja
Priority to EP10836074.4A priority patent/EP2511707B1/en
Priority to CN201080053485.0A priority patent/CN102667487B/zh
Publication of WO2011071155A1 publication Critical patent/WO2011071155A1/ja
Priority to US13/491,296 priority patent/US8486716B2/en
Priority to US13/916,274 priority patent/US20130295689A1/en
Priority to US15/380,220 priority patent/US10119980B2/en

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    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • 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
    • 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
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors

Definitions

  • the present invention relates to a method for detecting a basic peptide. More specifically, a basic peptide is detected by mixing a sample suspected of containing a basic peptide and a reagent containing denatured albumin, and detecting turbidity caused by the complex of the basic peptide and denatured albumin. Regarding the method.
  • the present invention also relates to a reagent for detecting a basic peptide used in the detection method.
  • ghrelin brain natriuretic peptide
  • ACTH adrenocorticotropic hormone
  • ANP atrial natriuretic peptide
  • bradykinin bradykinin
  • Peptides are considered useful as markers for diseases. For example, it is known that the plasma concentration of ghrelin, a basic peptide, decreases in patients with severe heart failure or gastric cancer with strong cachexia. Moreover, the blood concentration of BNP is important as a clinical index of heart failure, and BNP is used as a test marker.
  • These basic peptides are currently measured using immunological techniques such as enzyme immunoassay and electrochemiluminescence immunoassay.
  • immunological techniques such as enzyme immunoassay and electrochemiluminescence immunoassay.
  • measurement is performed using an antibody that specifically recognizes and binds to the basic peptide.
  • a step of binding a labeling substance to the complex is required, so that the operation becomes complicated.
  • Dennis et al. And Lowenthal et al. Reported a basic peptide detection method that can omit the step of binding a labeling substance, unlike the immunoassay method.
  • all of these methods are expensive and large-scale mass analyzers, surface plasmon resonance analyzers, and the like, so it is difficult to say that they are general-purpose and popular methods.
  • albumin is a protein contained in nature such as egg white, serum or milk. Taking serum albumin as an example, its physiological functions include regulating blood osmotic pressure, binding to blood metabolites such as fatty acids, hematin, and bilirubin, compounds such as drugs, and special peptides. Circulating the inside is known.
  • Baczynskyj et al. Suggest that bradykinin, also known as a blood hormone, may bind to BSA.
  • bovine serum albumin BSA
  • thermodynamic equilibrium theory is the cooperativity reflecting the dissociation constant (Kd value) indicating the strength (affinity) of binding and the allosteric structural change accompanying the binding between molecules actually generated in a solution. It is to describe with a clear numerical value (hill coefficient).
  • the detection method of Lowenthal et al. Is a mechanophysical method that relies on a mass analyzer.
  • a mixture of a basic peptide and unmodified albumin is once fixed and developed on a sample fixing polymer substrate of a mass spectrometer. That is, in this detection method, both the basic peptide and the native albumin are in a state of deviating from the actual solution equilibrium, and only a part of the molecular population is detected.
  • the inventors left the basic peptide and undenatured albumin in the solution all the time, and followed the natural binding by themselves, that is, the actual solution equilibrium. Unlike the above-described prior art, the present inventors examined a method for detecting a basic peptide or a complex of a basic peptide and native albumin using this solution equilibrium.
  • an object of the present invention is to provide a method capable of detecting a basic peptide easily and quickly without requiring a complicated operation such as binding of a labeling substance.
  • Another object of the present invention is to provide a reagent for detecting a basic peptide used in the detection method.
  • the present inventors mix a sample containing a basic peptide and a denatured bovine serum albumin solution, the denatured albumin and the basic peptide bind to form a complex, and the mixture becomes turbid. And that the degree of turbidity increases depending on the concentration of the basic peptide in the sample, thus completing the present invention.
  • the present invention (1) mixing a sample suspected of containing a basic peptide and a reagent containing denatured albumin to form a complex of the basic peptide and denatured albumin; (2) A method for detecting a basic peptide comprising the step of detecting the turbidity of the mixed solution obtained in step (1).
  • the present invention also provides a reagent for detecting a basic peptide containing denatured albumin.
  • a method capable of detecting a basic peptide easily and quickly without requiring complicated operations such as binding of a labeling substance and a reagent used in the method is provided.
  • FIG. 6 is a plot of OD measurement value ⁇ peptide concentration when ACTH partial peptide is detected using a BSA solution heat-denatured for 0, 5, 15, 60 and 120 minutes as a reagent.
  • FIG. 5 is a plot of OD measurement value ⁇ peptide concentration when ACTH partial peptide is detected in a sample containing nucleic acid and a sample not containing nucleic acid using the reagent of the present invention containing heat-denatured BSA.
  • FIG. 5 is a plot of OD measurement value ⁇ peptide concentration when an attempt is made to detect ⁇ -endorphin using the reagent of the present invention containing heat-denatured BSA. It is a plot of OD measurement value-peptide concentration when dynorphin A partial peptide is detected using the reagent of the present invention containing heat-denatured BSA.
  • FIG. 5 is a plot of OD measurement value ⁇ peptide concentration when a kininogen fragment is detected using the reagent of the present invention containing heat-denatured BSA.
  • FIG. 5 is a plot of OD measurement value ⁇ peptide concentration when a fragment of ITIH4 is detected using the reagent of the present invention containing heat-denatured BSA.
  • FIG. 6 is a plot of OD measurement value ⁇ peptide concentration when an attempt was made to detect fibrinogen ⁇ using the reagent of the present invention containing heat-denatured BSA.
  • FIG. 5 is a plot of OD measurement value ⁇ peptide concentration when C3f detection was attempted using the reagent of the present invention containing heat-denatured BSA.
  • FIG. 5 is a plot of OD measurement value ⁇ peptide concentration when an attempt was made to detect Factor XIII using the reagent of the present invention containing heat-denatured BSA.
  • FIG. 3 is a plot of OD measurement value ⁇ peptide concentration obtained by superimposing the plot of FIG. 1 and the plots of FIGS. 7-1 to 7-3. It is a plot of the fluorescence intensity-peptide concentration about the liquid mixture of the reagent of this invention containing heat-denatured BSA, and the sample containing ACTH partial peptide.
  • FIG. 3 is a plot of aggregate number-peptide concentration in a mixture of a reagent of the present invention containing heat-denatured BSA and a sample containing ACTH partial peptide.
  • FIG. 6 is a plot of OD measurement value ⁇ peptide concentration when ACTH partial peptide is detected in a sample containing serum and a sample not containing serum using the reagent of the present invention containing heat-denatured BSA.
  • the method of the present invention is a method for detecting a basic peptide comprising the following steps. (1) mixing a sample suspected of containing a basic peptide and a reagent containing denatured albumin to form a complex of the basic peptide and denatured albumin; (2) A step of detecting turbidity of the mixed solution obtained in the step (1).
  • the “basic peptide” means a short chain peptide having an isoelectric point (pI) within a basic range.
  • a peptide preferably has a pI of 8.0 or more, more preferably a pI of 8.5 or more, and a length of preferably 10 to 100 amino acids, more preferably 10 to 60 amino acids, still more preferably 10 to 40 amino acids. It is a peptide.
  • the peptide pI can be measured by a method known to those skilled in the art.
  • ExPASy Proteomics Server Compute Plot Param Tool http://expasy.ch/tools An algorithm or program such as (available from /protparam.html) can be used to determine the theoretical pI of a peptide.
  • the basic peptide to be detected is not particularly limited as long as it falls within the definition of the basic peptide described above, and may be a basic peptide of natural origin or a synthetic peptide. Also good.
  • the basic peptide to be detected is a basic peptide (for example, ACTH, ANP, BNP, and ghrelin, which is known to have a concentration in a specimen obtained from a living body having a specific disease state different from the normal concentration.
  • L-myc-1 proto-oncogene protein transcription factor SOX-3
  • fibrinogen alpha inter alpha trypsin inhibitor heavy chain subunit 4 (ITIH4)
  • Is alpha 2-HS-glycoprotein
  • prothrombin and kininogen Is also included.
  • a sample suspected of containing a basic peptide to be detected means a sample that may contain the basic peptide to be detected.
  • samples include specimens obtained from a living body, for example, blood (including whole blood, plasma, serum), urine, saliva, biological tissue extract, spinal fluid, pleural effusion, lymph, and the like.
  • the sample detected by the method of the present invention may contain impurities (for example, protein, nucleic acid, etc.) in addition to the basic peptide to be detected. In order to improve the detection sensitivity, these impurities are included. It is preferable that the sample has been previously removed, or a sample obtained by previously purifying and / or concentrating the basic peptide to be detected. Removal of such impurities and purification and concentration of the basic peptide can be performed by methods known to those skilled in the art.
  • impurities for example, protein, nucleic acid, etc.
  • the salt concentration in the sample is a salt concentration equivalent to 0 to 2 mS / cm in terms of electrical conductivity (for example, a potassium phosphate buffer solution of 10 ⁇ m or less or 10 ⁇ m or less. Tris-HCl) is preferable, and a salt concentration equivalent to 0 to 160 ⁇ S / cm in terms of electrical conductivity (for example, a potassium phosphate buffer solution of 900 ⁇ M or less) is more preferable.
  • albumin has the same meaning as a term generally known in the field of biology, and is a general term for a group of soluble proteins contained in cells and body fluids of animals and plants.
  • Known albumins of animal origin include serum albumin contained in serum, ovalbumin contained in egg white, lactalbumin contained in milk, and the like.
  • Known plant-derived albumins include leucosin contained in wheat and barley, legumelin contained in seeds such as peas and soybeans, and lysine contained in castor seeds.
  • a reagent containing denatured albumin preferably obtained from animal-derived albumin, more preferably serum albumin, ovalbumin or lactalbumin, more preferably serum albumin is used.
  • modified albumin means albumin in which a higher-order structure inherent in physiological conditions is destroyed due to a physical or chemical cause.
  • physical causes include treatment by heating, pressurization, freezing, ultrasonic crushing, and the like.
  • the chemical cause includes treatment with a denaturing agent, for example, a surfactant such as SDS, urea, guanidine hydrochloride and the like.
  • a reagent containing albumin denatured by such treatment preferably a reagent containing albumin denatured by heat, is used.
  • step (1) of the method of the present invention a sample suspected of containing a basic peptide to be detected and a reagent containing denatured albumin are mixed to form a complex of the basic peptide and denatured albumin.
  • the reagent and the sample are mixed so that the final concentration of the denatured albumin in the mixed solution of the reagent containing denatured albumin and the above sample is 0.2 to 0.6 w / v%, preferably 0.2 w / v%.
  • the mixing step is preferably performed at 4 to 50 ° C., more preferably 10 to 40 ° C.
  • the denatured albumin and the basic peptide are combined to form a complex. Furthermore, turbidity is generated in the mixed solution due to aggregation of the composites.
  • step (2) of the method of the present invention turbidity of the mixed solution obtained in the above step (1) is detected.
  • detecting turbidity is intended to include detecting the presence or absence of turbidity in a mixed solution and measuring the degree of turbidity (hereinafter referred to as “turbidity”). Detection of the presence or absence of turbidity can be performed by visual observation with the naked eye or a microscope or by using a turbidity measuring device. Turbidity can be measured using a turbidity measuring apparatus. Since the method of the present invention is a method for detecting turbidity caused by a complex of modified albumin and a basic peptide, a labeling substance and an antibody are not required as in the conventional method, and only a reagent containing modified albumin is used. Can be used to detect basic peptides.
  • Turbidity can be measured by irradiating light to the above mixture to obtain optical information.
  • optical information include absorbance, scattered light intensity, reflected light intensity, diffracted light intensity, fluorescence intensity, phosphorescence intensity, polarization state, refractive index, optical rotation, and the like.
  • absorbance is preferable because it can be measured with a simple apparatus.
  • a spectrophotometer UV-2500PC Shiladzu Corporation
  • the turbidity is preferably measured at a wavelength of 400 to 700 nm, more preferably 500 to 600 nm, which is the wavelength of visible light.
  • the method of the present invention can quantify the concentration of the basic peptide to be detected in the sample from the measured value of turbidity.
  • the concentration is quantified from a calibration curve prepared using a solution of a basic peptide to be detected whose concentration is known.
  • the above calibration curve can be created as follows. First, solutions of basic peptides to be detected at various concentrations (hereinafter referred to as “samples for preparing a calibration curve”) are prepared. Next, a sample for preparing a calibration curve and a reagent containing denatured albumin are mixed, and the turbidity of the obtained mixed solution is measured. The measured values are plotted for each sample for preparing a calibration curve, with the vertical axis representing the measured value of turbidity and the horizontal axis representing the concentration of the basic peptide.
  • the above-mentioned sample for preparing a calibration curve is a concentration until it can be confirmed that the measured turbidity has reached a plateau by gradually increasing the concentration from a solution not containing the basic peptide to be detected (blank solution).
  • ⁇ C n / (C n + Kd n ) (I)
  • Linearization formula: log ⁇ / (1- ⁇ ) ⁇ nlogC ⁇ nlogKd (II) (In the formula, ⁇ is a measured value of turbidity, C is a concentration of a basic peptide, Kd is a dissociation constant, and n is a Hill coefficient.)
  • the dissociation constant means the concentration of the peptide when the denatured albumin is saturated 50% with the basic peptide to be detected.
  • the Hill coefficient is an index of cooperativity in the binding (complex formation) between the basic peptide to be detected and denatured albumin.
  • the values of Kd and n are calculated from the measured values of the basic peptide concentration and turbidity of the calibration curve preparation sample.
  • the reason why turbidity is caused by mixing the above-described modified albumin and basic peptide is not completely clarified, but the following may be considered.
  • denatured albumin binds to a basic peptide having a positive charge by electrostatic interaction to form a complex.
  • turbidity arises because this complex aggregates.
  • bonding mode is an ionic bond. That the binding mode is ionic binding is also suggested by the fact that the method of the present invention improves the detection sensitivity of basic peptides under conditions of low salt concentration. This is because, under the condition of a low salt concentration, since there are few ions, it is considered that ion binding between the modified albumin and the basic peptide is promoted.
  • the binding between the denatured albumin and the basic peptide to be detected does not depend on the amino acid sequence and the three-dimensional structure of the peptide. Therefore, it is considered that the method of the present invention can be widely used for detection of various basic peptides.
  • the method of the present invention has a short period of time from mixing the reagent containing modified albumin and the sample containing basic peptide to the time when turbidity due to the complex of modified albumin and basic peptide occurs (for example, 20 seconds or less, preferably instantaneous). Therefore, the method of the present invention can rapidly detect a basic peptide contained in a sample.
  • the method of the present invention can also detect a basic peptide that is a disease marker contained in a sample obtained from a living body
  • the method of the present invention can also be used as a method for determining a specific disease. For example, when blood obtained from a subject is used as a specimen, a basic peptide to be detected, which is a specific disease marker, is first purified and / or concentrated from the blood, and then the concentration of the peptide is determined by the method of the present invention. It is possible to determine whether or not the subject suffers from the disease by comparing the obtained value with the normal value.
  • the “reagent containing modified albumin” used in the above-described method of the present invention is also one of the present invention. That is, the reagent of the present invention is a basic peptide detection reagent containing denatured albumin.
  • the reagent of the present invention can be obtained as follows.
  • an albumin solution is prepared by dissolving albumin of animal origin, preferably serum albumin, ovalbumin or lactalbumin, or a mixture thereof, more preferably serum albumin in a suitable solvent.
  • the albumin concentration of the solution is 0.2 to 5.0 w / v%, preferably 0.2 to 0.6 w / v%.
  • the solvent is not particularly limited as long as it does not interfere with the complex formation between the modified albumin and the basic peptide.
  • ultrapure water 10 mM Tris-HCl (pH 5.5 to 7.5), 10 mM potassium phosphate.
  • the buffer solution include a buffer solution (pH 5.5 to 7.5).
  • ultrapure water is preferable.
  • “ultra pure water” means water having a specific resistance value at 25 ° C. of 18 M ⁇ ⁇ cm or more.
  • Milli-Q (registered trademark) water is preferable.
  • the obtained albumin solution is subjected to physical denaturation treatment or chemical denaturation treatment.
  • physical denaturation treatment include heating, pressurization, freezing, and ultrasonic crushing.
  • chemical denaturation treatment include treatment with a surfactant such as SDS, a denaturant such as urea, guanidine hydrochloride, or a mixture thereof.
  • the above albumin solution is heated at a temperature of 100 to 130 ° C., preferably 110 to 120 ° C., for 5 to 120 minutes, preferably 10 to 60 minutes.
  • a method for denaturation using a mixture of SDS and urea include, for example, a method in which SDS (final concentration 1 to 30 w / v%) and urea (final concentration 500 mM to 5 M) are added to the albumin solution and mixed. Is mentioned.
  • the reagent of the present invention is preferably a reagent containing albumin modified by heat or albumin modified by a mixture of SDS and urea, more preferably a reagent containing albumin modified by heat.
  • the reagent of the present invention is preferably in the form of a solution.
  • the modified albumin and the basic peptide form a complex via an ionic bond. It has also been found that the relationship between the turbidity produced by the complex and the concentration of the basic peptide follows the Hill formula of the above formula (I). Therefore, the structure of the modified albumin contained in the reagent of the present invention is not clear in detail, but it has a plurality of sites that electrostatically bind to the basic peptide, and is positively cooperating with the basic peptide. Is considered to be an allosteric protein.
  • the reagent of the present invention preferably contains denatured albumin that binds with a basic peptide having the amino acid sequence shown in SEQ ID NO: 1 with a dissociation constant of 5 to 220 ⁇ M.
  • the reagent of the present invention preferably contains a basic peptide having the amino acid sequence set forth in SEQ ID NO: 1 and denatured albumin that binds with a Hill coefficient greater than 1 and less than 11.
  • the dissociation constant and Hill coefficient can be determined from the Hill equation of the above formula (I) if the measured value of turbidity and the concentration of the basic peptide are known.
  • the amino acid sequence described in SEQ ID NO: 1 is a sequence at positions 1 to 24 on the N-terminal side of ACTH.
  • Example 1 Detection of Basic Peptide Using Modified BSA and Native BSA Experimental method (1) Preparation of denatured BSA solution and non-denatured BSA solution as detection reagents BSA (Sigma-Aldrich) was dissolved in Milli-Q (registered trademark) water (Millipore) and BSA solution ( 1.0 w / v%) was prepared. This BSA solution was divided into two equal parts, one being an unmodified BSA solution and the other being heated at 110 ° C. for 15 minutes by an autoclave to obtain a solution of heat-modified BSA (hereinafter also referred to as “heat-modified BSA”). .
  • BSA heat-modified BSA
  • sample containing basic peptide to be detected As a basic peptide to be detected, an ACTH partial peptide consisting of amino acids 1 to 24 of ACTH (Peptide Institute, Inc.) was used. This peptide was dissolved in ultrapure water to prepare a sample (1.0 mg / ml) containing the basic peptide to be detected.
  • heat-denatured BSA When comparing the Kd values of heat-denatured BSA and unmodified BSA, heat-denatured BSA showed a lower value. This shows that heat-denatured BSA has a much higher affinity for ACTH, which is a basic peptide, than native BSA.
  • sample containing basic peptide to be detected As a basic peptide to be detected, an ACTH partial peptide consisting of amino acids 1 to 24 of ACTH (Peptide Institute, Inc.) was used. This peptide was dissolved in ultrapure water to prepare a sample (1.0 mg / ml) containing the basic peptide to be detected.
  • Example 3 Examination of influence of nucleic acid in detection of basic peptide Experimental Method (1) Preparation of Denatured BSA Solution as Detection Reagent A heat-denatured BSA solution was prepared in the same manner as in Example 1 above.
  • sample containing basic peptide to be detected As a basic peptide to be detected, an ACTH partial peptide consisting of amino acids 1 to 24 of ACTH (Peptide Institute, Inc.) was used. This peptide was dissolved in ultrapure water to prepare a sample (1.0 mg / ml) containing the basic peptide to be detected.
  • Example 4 Detection of basic peptide and neutral peptide Experimental Method (1) Preparation of Denatured BSA Solution as Detection Reagent A heat-denatured BSA solution was prepared in the same manner as in Example 1 above.
  • the method of the present invention can be specifically used for the detection of the presence of a basic peptide and the quantification of its concentration.
  • Example 5 Detection of basic peptide as cancer metastasis marker Experimental Method (1) Preparation of Denatured BSA Solution as Detection Reagent A heat-denatured BSA solution was prepared in the same manner as in Example 1 above.
  • sample containing basic peptide to be detected As a basic peptide to be detected, a fragment of kininogen known as a cancer metastasis marker (positions 439 to 457 (SEQ ID NO: 4); synthesized by Bilodika Corporation) and A fragment of ITIH4 (positions 611 to 642 (SEQ ID NO: 5); synthesized by Virodica Co., Ltd.) was used. These peptides were dissolved in ultrapure water to prepare samples (1.0 mg / ml) containing the respective peptides.
  • a cancer metastasis marker positions 439 to 457 (SEQ ID NO: 4); synthesized by Bilodika Corporation
  • ITIH4 positions 611 to 642 (SEQ ID NO: 5); synthesized by Virodica Co., Ltd.
  • the method of the present invention can be used for the detection of the presence of a basic peptide that is a cancer marker and the quantification of its concentration.
  • Example 6 Detection of basic peptides using heat-denatured human serum albumin (HSA) and heat-denatured ovalbumin (OA) Experimental Method (1) Preparation of Modified HSA Solution and Modified OA Solution as Detection Reagent HSA (Wako) was dissolved in ultrapure water to prepare an HSA solution (1.0 w / v%). This HSA solution was heated in an autoclave at 115 ° C. for 15 minutes to obtain a solution of heat-modified HSA (hereinafter also referred to as “heat-modified HSA”). OA (Worthington) was dissolved in ultrapure water to prepare an OA solution (1.0 w / v%).
  • HSA heat-denatured human serum albumin
  • OA ovalbumin
  • heat-denatured OA a solution of heat-denatured OA (hereinafter also referred to as “heat-denatured OA”). Obtained.
  • Example 7 Examination of concentration of detection reagent in peptide detection Experimental method (1) Preparation of denatured BSA solution as detection reagent A BSA solution (5.0 w / v%) was prepared by dissolving in BSA (Sigma-Aldrich) ultrapure water. This BSA solution was heated in an autoclave at 115 ° C. for 15 minutes to obtain a heat-denatured BSA solution.
  • BSA Sigma-Aldrich
  • Example 8 Evaluation of detectability for neutral peptides and acidic peptides using a modified BSA solution Experimental Method (1) Preparation of Denatured BSA Solution as Detection Reagent A heat-denatured BSA solution was prepared in the same manner as in Example 1 above.
  • FIG. 7-4 a graph combining the above result and the result of detecting the basic peptide of Example 1 is shown in FIG. 7-4. As is apparent from this graph, it can be seen that the method of the present invention does not have the ability to detect acidic peptides or neutral peptides, but exhibits specific detection ability only for basic peptides.
  • Example 9 Examination of peptide concentration limit (LoD) in peptide detection of heat-denatured BSA solution Experimental Method (1) Preparation of Denatured BSA Solution as Detection Reagent A heat-denatured BSA solution was prepared in the same manner as in Example 1 above.
  • Example 10 Evaluation of Detectability for Basic Peptides in Samples Containing Serum Experimental Method (1) Preparation of Denatured BSA Solution as Detection Reagent A heat-denatured BSA solution was prepared in the same manner as in Example 1 above.
  • ACTH partial peptide consisting of amino acids 1 to 24 of ACTH (Biologica Co., Ltd.) was used as the detection target basic peptide. After dissolving this peptide in ultrapure water, it was mixed with the serum (SUNFCO) of a healthy US person (female, 16 age) to prepare a sample containing the basic peptide to be detected and serum. In this sample, the ACTH partial peptide concentration and the serum concentration were adjusted to the final concentrations shown in the following (3).
  • FIG. 10 shows that the sample is not a pure sample containing only the basic peptide to be detected as in Examples 1 to 9, but a sample containing serum such as an actual sample collected from the subject. It was revealed that the sex peptide could be detected. Further, in FIG. 10, when the concentrations of the ACTH partial peptide in the serum solution and the control solution corresponding to a predetermined OD value are compared, it is found that the amount of ACTH partial peptide in the serum solution is about twice that of the control solution. This is considered to be the influence of competitive inhibition by serum HSA.
  • a process of concentrating the peptide can be added as a pretreatment.
  • the process is shown in the following example.
  • Example 11 Concentration process of basic peptide in sample containing serum and detection of concentrated basic peptide Experimental Method (1) Preparation of Denatured BSA Solution as Detection Reagent A heat-denatured BSA solution was prepared in the same manner as in Example 1 above.
  • ACTH partial peptide consisting of amino acids 1 to 24 of ACTH (Biologica Co., Ltd.) was used as the detection target basic peptide. This peptide was dissolved in ultrapure water, and then mixed with serum from a healthy US person (female, 16age) (SUNFCO) to prepare a real sample serum sample containing a basic peptide to be detected. In this sample, the concentration of ACTH partial peptide and the serum concentration were adjusted to the following concentrations (3).
  • sample solution in which basic peptide to be detected is concentrated 6 ml of Glycerol solution (Agilent technologies) and 600 ⁇ l of OFFGEL Buffer (Agilent technologies) are mixed, and then ultrapure water is added to make a total volume of 50 ml A -10 peptide buffer solution (x1.25) was prepared.
  • This solution was mixed with the sample of (2) above to prepare an ACTH partial peptide concentration of 20 ⁇ M and a 1000-fold diluted peptide buffer solution ( ⁇ 1) of serum.
  • ⁇ 1 Using this solution and a High Resolution IPG gel for pH 3-10 Fraction, fractionation was performed with an OFFGEL fractionator (Agilent technologies). Thereafter, Fraction containing ACTH partial peptide (solution in contact with the cathode part) was collected, and a solution added with 100 ⁇ l of ultrapure water was used as a basic peptide concentrated sample.
  • the method of the present invention can detect a very small amount of a basic peptide in an actual sample by concentrating the basic peptide with an OFFGEL fractionator as a pretreatment.

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PCT/JP2010/072256 2009-12-10 2010-12-10 塩基性ペプチドの検出方法および塩基性ペプチド検出用試薬 WO2011071155A1 (ja)

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EP10836074.4A EP2511707B1 (en) 2009-12-10 2010-12-10 Method for detection of basic peptide and reagent for detection of basic peptide
CN201080053485.0A CN102667487B (zh) 2009-12-10 2010-12-10 碱性肽的检测方法及碱性肽检测用试剂
US13/491,296 US8486716B2 (en) 2009-12-10 2012-06-07 Method for detection of basic peptide and reagent for detection of basic peptide
US13/916,274 US20130295689A1 (en) 2009-12-10 2013-06-12 Method for detection of basic peptide and reagent for detection of basic peptide
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JP6675167B2 (ja) 2015-08-28 2020-04-01 シスメックス株式会社 ペプチドの遊離方法及び回収方法、並びにペプチド遊離剤及び試薬キット
CN110746485A (zh) * 2019-10-09 2020-02-04 天津科技大学 新型碱性蛋白酶抑制肽的筛选
CN111138513B (zh) * 2020-01-06 2022-10-18 天津科技大学 谷氨酰胺转氨酶交联肽的筛选

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