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WO2011078307A1 - Procédé pour mesurer la substance p dans un échantillon d'urine - Google Patents

Procédé pour mesurer la substance p dans un échantillon d'urine Download PDF

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Publication number
WO2011078307A1
WO2011078307A1 PCT/JP2010/073285 JP2010073285W WO2011078307A1 WO 2011078307 A1 WO2011078307 A1 WO 2011078307A1 JP 2010073285 W JP2010073285 W JP 2010073285W WO 2011078307 A1 WO2011078307 A1 WO 2011078307A1
Authority
WO
WIPO (PCT)
Prior art keywords
urine
substance
urine sample
measurement
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2010/073285
Other languages
English (en)
Japanese (ja)
Inventor
一彦 佐々木
典仁 青山
修 草田
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Minaris Medical Co Ltd
Original Assignee
Kyowa Medex Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyowa Medex Co Ltd filed Critical Kyowa Medex Co Ltd
Priority to JP2011547638A priority Critical patent/JP5663494B2/ja
Publication of WO2011078307A1 publication Critical patent/WO2011078307A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/493—Physical analysis of biological material of liquid biological material 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

Definitions

  • the present invention relates to a method for measuring substance P useful as a marker for inflammation, allergic diseases and the like.
  • urine Unlike whole blood, serum and plasma, urine has the advantage that it can be collected without imposing a burden on the subject, while bacteria are easy to propagate and are susceptible to proteolytic enzymes. , Have the disadvantages. Therefore, when 24-hour urine storage is used as a specimen or when it is stored for a long period of time, it is often performed to stabilize a component to be measured in urine by coexisting a preservative.
  • a preservative protease inhibitors, hydrochloric acid, toluene, xylene, sodium azide, chelating agents, albumin, isothiazolone compounds, buffers, surfactants, and combinations of these elements are known (for example, (See Patent Documents 1 to 4).
  • the preservative may affect the measurement of the component to be measured, sufficient management is required when urine is used as a specimen, and urine has a difficult aspect of being used as a specimen.
  • the collected urine itself (original urine) is buffered to avoid the influence of biological interfering substances present in urine and the above preservatives.
  • highly diluted with an aqueous medium such as, and highly diluted urine is subjected to measurement.
  • the method of measuring the measurement target component by diluting the raw urine has a different measurement target component concentration for each urine sample, and it is necessary to set an appropriate dilution ratio for each urine sample.
  • the urine is highly diluted, so that the concentration of the component to be measured is significantly reduced, and the accuracy and reliability of the measurement are significantly reduced.
  • Substance P is a peptide consisting of 11 amino acid sequences.
  • Substance P is a chemical transmitter of the primary sensory nerve, and the presence of this substance in a high concentration in the body is suspected to increase pain and inflammation.
  • Substance P is attracting attention as a useful marker of inflammation and allergic diseases.
  • Non-Patent Document 1 As a method for measuring substance P in a specimen, a method in which serum is acid-treated, extracted with acetone and ether, and further purified by HPLC using a reverse phase column is then measured by enzyme immunoassay. (See Non-Patent Document 1). However, since this method is very complicated to operate and the chemical treatment conditions are harsh, it does not give accurate measurement values.
  • An object of the present invention is to provide a method for easily and accurately measuring substance P in a urine sample.
  • the present inventors have not heated the sample when measuring the peptide in the sample because the peptide is usually denatured by heat treatment.
  • the substance P the finding that it can be accurately measured by heat-treating a urine sample was found, and the present invention was completed. That is, the present invention relates to the following (1) to (5).
  • a method for measuring substance P in a urine sample wherein the urine sample is subjected to heat treatment, and the substance P in the urine sample after the heat treatment is measured.
  • the measurement method according to (1), wherein the heat treatment is a treatment of heating the urine sample at 30 to 55 ° C.
  • the measurement method according to (1) or (2), wherein the heat treatment is a treatment of heating a urine sample at 35 to 50 ° C. for 15 minutes to 4 hours.
  • the present invention provides a method for easily and accurately measuring substance P in a urine sample.
  • the urine sample in the present invention is not particularly limited as long as it is a urine sample containing substance P, and examples thereof include urine collected from animals.
  • animals include humans, monkeys, apes (gorillas, orangutans, chimpanzees, long-haired), dogs, cats, rats, mice, rabbits, and the like.
  • an additive such as a preservative to raw urine
  • the preservative include known preservatives such as protease inhibitors, hydrochloric acid, toluene, sodium azide and the like.
  • the heat treatment of the urine sample in the present invention is not particularly limited as long as the substance P in the urine sample can be measured, and includes, for example, a treatment of heating urine at 30 to 55 ° C., such as 35 to 50 ° C. A heating process is preferred.
  • the heating time of the urine sample in the heat treatment is not particularly limited as long as the substance P in the urine sample can be measured, and is, for example, 10 minutes to 6 hours. In particular, when the heating temperature is 35 to 50 ° C., 15 minutes to 4 hours are preferable, and 1 hour to 3 hours are particularly preferable.
  • a refrigeration treatment, a freezing treatment, a storage treatment or the like may be performed before and after the heat treatment. Further, the heat treatment may be performed a plurality of times.
  • the refrigeration process include a process in which a urine sample is refrigerated at ⁇ 20 to 0 ° C.
  • the freezing process include a process of freezing a urine sample at ⁇ 80 to ⁇ 20 ° C.
  • the storage process include a process of leaving a urine sample at 4 to 25 ° C. for 2 hours or more.
  • the method for measuring substance P in a urine sample of the present invention is a method using a heat-treated urine sample, and can be specifically performed by the following steps. [1] A step of heat-treating a urine sample; [2] a step of measuring substance P in the urine sample using the urine sample heat-treated in [1]; and [3] A calibration curve created using a substance P having a known concentration in advance and representing the relationship between the concentration of substance P and the amount of information is compared with the measurement value obtained in the measurement in [2]. Determining the concentration of substance P in the urine sample;
  • the measurement of substance P in a urine sample can be performed by any method as long as it can measure substance P.
  • it can be performed by a known method such as an immunological measurement method.
  • the measurement of substance P in a urine sample can also be performed using a commercially available kit.
  • the immunological measurement method uses a reaction (antigen-antibody reaction) between a measurement target component and an antibody or a fragment thereof that binds to the measurement target component.
  • the sandwich method, competition method, enzyme immunoassay method, chemiluminescent enzyme Examples include immunoassay, fluorescence immunoassay, electrochemiluminescence immunoassay, and radioimmunoassay.
  • Commercially available kits include “Substance P EIA Kit” (Cayman Chemical), “Substance P EIA Kit” (Assay Designs), “DRG Human Substance P ELISA (EIA-3120)” (DRG International) Etc.
  • the package insert of “Substance P EIA Kit” (manufactured by Cayman Chemical) describes a range of 8.2 to 500 pg / mL as a measurement range of measurement using the kit.
  • the substance P concentration in a urine sample was similarly determined using a urine sample obtained by heat-treating raw urine at 40 ° C. for 60 minutes.
  • the measurement results are shown in Table 1.
  • the correction value is a value calculated by multiplying the actual measurement value by the dilution factor.
  • the measurement value in the measurement using raw urine without heat treatment is lower than the correction value in the measurement using diluted urine. Indicated.
  • the correction value at the 2-fold dilution almost coincided with the measured value using the heat-treated raw urine.
  • the concentration of substance P in diluted urine was very low in urine diluted 8 times and was outside the measurement range of the kit used, so accurate measurement could not be performed.
  • the correction value at the 8-fold dilution almost coincided with the measurement value obtained using the heat-treated raw urine.
  • the correction value at the 8-fold dilution almost coincided with the measured value using the heat-treated raw urine.
  • the substance P in the urine sample can be measured easily and accurately without complicated operations.
  • Each of the prepared samples for addition recovery test (1) to (3) was heat-treated at 40 ° C. for 20 to 120 minutes, and the substance P in each sample after the heat treatment was replaced with a substance P measurement kit “Substance P EIA Using “Kit” (manufactured by Cayman® Chemical), measurement was performed according to the method described in the package insert of the kit.
  • a urine sample (original urine) collected from a healthy person is heated at 30 ° C, 35 ° C, 40 ° C, 45 ° C, 50 ° C and 55 ° C, and then the heat-treated urine Using the sample, substance P in the urine sample was measured using a substance P measurement kit “Substance P EIA Kit” (manufactured by Cayman Chemical) according to the method described in the package insert of the kit. The measurement results are shown in Table 3.
  • the numerical values in Table 3 represent relative values (%) when the measurement value in the measurement using a urine sample heat-treated at 40 ° C. for 60 minutes is taken as 100. From Table 3, the heating time at which accurate measurement is possible differs depending on the heating temperature of the urine sample. The higher the heating temperature, the shorter the heating time and the lower the measured value in the long-time heating. It has been found.
  • the measured value in Table 4 means a relative value (%) when the measured value in a measurement (condition 3) using a urine sample heat-treated at 40 ° C. for 60 minutes is taken as 100.
  • Table 4 shows that when a heat-treated urine sample (conditions 3-6, 9-10) is used, substance P in the urine sample can be accurately measured, whereas urine not heat-treated When specimens (conditions 1 and 2, 7 to 8) were used, the measured values were remarkably low, and it was found that substance P in urine specimens could not be measured accurately with these urine specimens. From this result, it was found that the substance P in the urine sample can be accurately measured by heat-treating the urine sample.
  • the present invention provides a simple and accurate method for measuring substance P in a urine sample, which is useful for diagnosing inflammation and allergic diseases.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé permettant de mesurer facilement et précisément la substance P dans un échantillon d'urine. Elle concerne spécifiquement un procédé permettant de mesurer la substance P dans un échantillon d'urine qui est caractérisé en ce que l'échantillon d'urine est soumis à un traitement thermique et en ce que la substance P dans l'échantillon d'urine traité thermiquement est mesurée. À ce sujet, le traitement thermique est un traitement dans lequel un échantillon d'urine est chauffé à 30-55 °C, de préférence un traitement dans lequel un échantillon d'urine est chauffé à 35-55 °C pendant 15 minutes à 4 heures. Par ce procédé, la substance P dans un échantillon d'urine peut être mesurée facilement et précisément.
PCT/JP2010/073285 2009-12-25 2010-12-24 Procédé pour mesurer la substance p dans un échantillon d'urine Ceased WO2011078307A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2011547638A JP5663494B2 (ja) 2009-12-25 2010-12-24 尿検体中のサブスタンスｐの測定方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2009295509 2009-12-25
JP2009-295509 2009-12-25

Publications (1)

Publication Number Publication Date
WO2011078307A1 true WO2011078307A1 (fr) 2011-06-30

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PCT/JP2010/073285 Ceased WO2011078307A1 (fr) 2009-12-25 2010-12-24 Procédé pour mesurer la substance p dans un échantillon d'urine

Country Status (2)

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JP (1) JP5663494B2 (fr)
WO (1) WO2011078307A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021090901A1 (fr) * 2019-11-05 2021-05-14 国立大学法人 東京大学 Procédé d'évaluation de maladies inflammatoires chez des animaux de la famille des félins
US20220137052A1 (en) * 2019-07-22 2022-05-05 Roche Diagnostics Operations, Inc. Substance p as blood biomarker for the non-invasive diagnosis of endometriosis

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1160600A (ja) * 1997-08-22 1999-03-02 Eiken Chem Co Ltd 尿中ミオグロビンの安定化方法、安定化剤およびこれを応用した免疫学的測定方法
JP2006508357A (ja) * 2002-11-28 2006-03-09 コミサリア ア レネルジィ アトミーク アナライトを連続的に検出するための方法、使用される３官能性検出試薬及び検出デバイス
WO2007010995A1 (fr) * 2005-07-20 2007-01-25 Kyowa Medex Co., Ltd. Procédé pour stabiliser un peptide contenu dans un échantillon biologique
WO2008022177A2 (fr) * 2006-08-15 2008-02-21 Prometheus Laboratories, Inc. Procédés de diagnostic de syndrome de côlon irritable
JP2008176530A (ja) * 2007-01-18 2008-07-31 Oki Electric Ind Co Ltd 窓口業務支援システム

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5162751B2 (ja) * 2007-01-17 2013-03-13 国立大学法人 宮崎大学 急性腎不全の検出方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1160600A (ja) * 1997-08-22 1999-03-02 Eiken Chem Co Ltd 尿中ミオグロビンの安定化方法、安定化剤およびこれを応用した免疫学的測定方法
JP2006508357A (ja) * 2002-11-28 2006-03-09 コミサリア ア レネルジィ アトミーク アナライトを連続的に検出するための方法、使用される３官能性検出試薬及び検出デバイス
WO2007010995A1 (fr) * 2005-07-20 2007-01-25 Kyowa Medex Co., Ltd. Procédé pour stabiliser un peptide contenu dans un échantillon biologique
WO2008022177A2 (fr) * 2006-08-15 2008-02-21 Prometheus Laboratories, Inc. Procédés de diagnostic de syndrome de côlon irritable
JP2008176530A (ja) * 2007-01-18 2008-07-31 Oki Electric Ind Co Ltd 窓口業務支援システム

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TETSUO YAMADA: "Kanshitsusei Bokoen to mast cell", VOIDING DISORDERS DIGEST, vol. 12, no. 1, 10 March 2004 (2004-03-10), pages 39 - 44 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220137052A1 (en) * 2019-07-22 2022-05-05 Roche Diagnostics Operations, Inc. Substance p as blood biomarker for the non-invasive diagnosis of endometriosis
WO2021090901A1 (fr) * 2019-11-05 2021-05-14 国立大学法人 東京大学 Procédé d'évaluation de maladies inflammatoires chez des animaux de la famille des félins

Also Published As

Publication number Publication date
JP5663494B2 (ja) 2015-02-04
JPWO2011078307A1 (ja) 2013-05-09

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