WO2011078307A1 - Method for measuring substance p in urine sample - Google Patents

Method for measuring substance p in urine sample Download PDF

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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
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urine
substance
urine sample
measurement
sample
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PCT/JP2010/073285
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French (fr)
Japanese (ja)
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一彦 佐々木
典仁 青山
修 草田
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協和メデックス株式会社
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Priority to JP2011547638A priority Critical patent/JP5663494B2/en
Publication of WO2011078307A1 publication Critical patent/WO2011078307A1/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
    • G01N33/483—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.

Abstract

Disclosed is a method for easily and accurately measuring substance P in a urine sample. Specifically disclosed is a method for measuring substance P in a urine sample, which is characterized in that the urine sample is subjected to heat treatment and the substance P in the heat-treated urine sample is measured. In this connection, the heat treatment is treatment in which a urine sample is heated at 30-55˚C, preferably treatment in which a urine sample is heated at 35-50˚C for 15 minutes to 4 hours. By this method, substance P in a urine sample can be measured easily and accurately.

Description

尿検体中のサブスタンスＰの測定方法Method for measuring substance P in urine samples
　本発明は、炎症、アレルギー疾患等のマーカーとして有用なサブスタンスＰの測定方法に関する。 The present invention relates to a method for measuring substance P useful as a marker for inflammation, allergic diseases and the like.
　臨床診断において、尿を検体として用いる尿中の測定対象成分の測定と、その測定に基づく疾患の診断がしばしば行われている。例えば、尿試験紙を用いた尿蛋白、尿潜血、尿中ヘモグロビン、尿糖等の検査は日常的に行われている。また、尿中の蛋白質及びペプチドは、腎臓、泌尿器系、循環器系等の疾患及び状態のマーカーとして有用であり、例えばＣ－ペプチド、Ｌ型脂肪酸結合蛋白（Ｌ－ＦＡＢＰ）等が尿を検体として用いて測定されている。 In clinical diagnosis, measurement of a component to be measured in urine using urine as a specimen and diagnosis of a disease based on the measurement are often performed. For example, tests for urine protein, urine occult blood, urinary hemoglobin, urine sugar, and the like using urine test paper are routinely performed. In addition, proteins and peptides in urine are useful as markers for diseases and conditions of kidney, urinary system, circulatory system, etc. For example, C-peptide, L-type fatty acid binding protein (L-FABP), etc. Measured using as
　尿は、全血、血清や血漿とは異なり、被検者へ負担を掛けることなく採取することができる、という利点を有する一方、細菌が繁殖し易く、また、蛋白質分解酵素の影響を受け易い、という欠点を有する。従って、２４時間蓄尿を検体として用いる場合や、長期間保存する場合には、保存剤を共存させて、尿中の測定対象成分を安定化させることがしばしば行われる。保存剤として、プロテアーゼ阻害剤、塩酸、トルエン、キシレン、アジ化ナトリウム、キレート剤、アルブミン、イソチアゾロン系化合物、緩衝剤、界面活性剤の他、これらの要素の組み合わせ等が知られている（例えば、特許文献１～４参照）。しかしながら、保存剤が測定対象成分の測定に影響することもあり、尿を検体として用いる場合には充分な管理が必要であり、尿は検体として使い辛い面を有している。 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. As preservatives, 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). However, since 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.
　また、従来、尿中の測定対象成分を測定するに際しては、尿中に存在する生体由来の妨害物質や上記の保存剤の影響を回避するために、採取した尿そのもの（原尿）を緩衝液等の水性媒体で高度に希釈し、高度に希釈された尿を測定に供することもしばしば行われる。しかしながら、原尿を希釈して、測定対象成分を測定する方法は、測定対象成分濃度が尿検体毎に異なり、尿検体毎に適切な希釈率を設定する必要があり、操作が煩雑になると共に、尿を高度に希釈することにより、測定対象成分の濃度が著しく低下し、測定の正確性と信頼性が著しい低下する、という欠点を有している。 Conventionally, when measuring a component to be measured in urine, the collected urine itself (original urine) is buffered to avoid the influence of biological interfering substances present in urine and the above preservatives. Often, highly diluted with an aqueous medium such as, and highly diluted urine is subjected to measurement. However, 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.
　サブスタンスＰは、１１個のアミノ酸配列からなるペプチドである。サブスタンスＰは、第一次知覚神経の化学伝達物質であり、この物質が体内に高濃度で存在することは、痛みや炎症の神経伝達亢進が疑われる。サブスタンスＰは、炎症、アレルギー疾患の有用なマーカーとして注目を集めている。 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.
　検体中のサブスタンスＰ測定方法としては、これまで、血清を酸処理した後、アセトンおよびエーテルで抽出し、さらに逆相カラムによるＨＰＬＣで精製したサブスタンスＰを酵素免疫測定法で測定する方法が知られている（非特許文献１参照）。しかしながら、本方法は操作が非常に煩雑で、かつ、化学的処理条件が過酷なため、正確な測定値を与えるものではなかった。 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.
　また、採血後の血液中サブスタンスＰは非常に不安定であるため、正確な測定値を得ることは困難であったが、採血後の血液に糖類を添加することにより、採血後の血液中のサブスタンスＰが安定化され、本安定化方法を利用した血液中のサブスタンスＰの正確な測定方法が報告されている（特許文献５参照）。 Moreover, since the substance P in the blood after blood collection is very unstable, it was difficult to obtain an accurate measurement value. However, by adding saccharide to the blood after blood collection, Substance P has been stabilized, and an accurate method for measuring substance P in blood using this stabilization method has been reported (see Patent Document 5).
　しかしながら、この方法も検体として血液を用いることから、被検者に採血という負担を強いることになる。 However, since this method also uses blood as a specimen, it places a burden on the subject to collect blood.
特表平１０－５０７２６９号公報Japanese National Patent Publication No. 10-507269 特開平１０－２８２０９５号公報Japanese Patent Laid-Open No. 10-282095 特開２０００－３５２５６５号公報JP 2000-352565 A 国際公開第２００９／０４１５７７号パンフレットInternational Publication No. 2009/041577 Pamphlet 国際公開第２００７／０１０９９５号パンフレットInternational Publication No. 2007/010995 Pamphlet
　本発明の目的は、尿検体中のサブスタンスＰを簡便、かつ、正確に測定するための方法を提供することにある。 An object of the present invention is to provide a method for easily and accurately measuring substance P in a urine sample.
　本発明者らは、上記課題を解決すべく鋭意検討した結果、ペプチドは通常、加熱処理によって変性を受けるため、検体中のペプチドの測定に際して、検体を加熱することは行わないが、尿検体中のサブスタンスＰについては、尿検体を加熱処理することによって、正確に測定できる、という知見を見出し、本発明を完成させた。すなわち、本発明は以下の（１）～（５）に関する。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have not heated the sample when measuring the peptide in the sample because the peptide is usually denatured by heat treatment. As for 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).
（１）　尿検体を加熱処理し、加熱処理後の尿検体中のサブスタンスＰを測定することを特徴とする、尿検体中のサブスタンスＰの測定方法。
（２）　加熱処理が、尿検体を３０～５５℃で加熱する処理である、（１）記載の測定方法。
（３）　加熱処理が、尿検体を３５～５０℃で、１５分間～４時間加熱する処理である、（１）または（２）記載の測定方法。
（４）　サブスタンスＰの測定が、免疫学的測定法により行われる、（１）～（３）のいずれかに記載の測定方法。
（５）　尿検体が、原尿である、（１）～（４）のいずれかに記載の測定方法。 (1) 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.
(2) The measurement method according to (1), wherein the heat treatment is a treatment of heating the urine sample at 30 to 55 ° C.
(3) 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.
(4) The measurement method according to any one of (1) to (3), wherein the substance P is measured by an immunological measurement method.
(5) The measurement method according to any one of (1) to (4), wherein the urine sample is raw urine.
　本発明により、尿検体中のサブスタンスＰを簡便、かつ、正確に測定するための方法が提供される。 The present invention provides a method for easily and accurately measuring substance P in a urine sample.
　本発明における尿検体は、サブスタンスＰを含む尿検体であれば特に制限はなく、例えば動物より採取された尿等が挙げられる。動物としては、例えばヒト、猿、類人猿（ゴリラ、オランウータン、チンパンジー、てながざる）、イヌ、ネコ、ラット、マウス、ウサギ等が挙げられる。本発明においては、原尿のみならず、原尿に保存剤等の添加物が添加された尿等も用いることができるが、原尿を用いることが好ましい。保存剤としては、例えばプロテアーゼインヒビター、塩酸、トルエン、アジ化ナトリウム等の公知の保存剤等が挙げられる。 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. Examples of animals include humans, monkeys, apes (gorillas, orangutans, chimpanzees, long-haired), dogs, cats, rats, mice, rabbits, and the like. In the present invention, not only raw urine but also urine obtained by adding an additive such as a preservative to raw urine can be used, but it is preferable to use raw urine. Examples of the preservative include known preservatives such as protease inhibitors, hydrochloric acid, toluene, sodium azide and the like.
　本発明における尿検体の加熱処理は、尿検体中のサブスタンスＰの測定を可能とする範囲であれば特に制限はなく、例えば尿を３０～５５℃で加熱する処理が挙げられ、３５～５０℃で加熱する処理が好ましい。加熱処理における尿検体の加熱時間は、尿検体中のサブスタンスＰの測定を可能とする範囲であれば特に制限はなく、例えば１０分間～６時間である。特に、加熱温度が３５～５０℃であれば、１５分間～４時間が好ましく、１時間～３時間が特に好ましい。 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.
　本発明においては、尿検体を少なくとも１回加熱処理すれば、加熱処理の前後に冷蔵処理、冷凍処理または保存処理等の処理を行ってもよい。また、加熱処理は複数回行ってもよい。冷蔵処理としては、例えば尿検体を－２０～０℃で冷蔵する処理等が挙げられる。冷凍処理としては、例えば尿検体を－８０～－２０℃で冷凍する処理等が挙げられる。保存処理としては、例えば尿検体を４～２５℃で、２時間以上放置する処理等が挙げられる。 In the present invention, if the urine sample is heat-treated at least once, 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. Examples of the refrigeration process include a process in which a urine sample is refrigerated at −20 to 0 ° C. Examples of the freezing process include a process of freezing a urine sample at −80 to −20 ° C. Examples of the storage process include a process of leaving a urine sample at 4 to 25 ° C. for 2 hours or more.
　本発明の尿検体中のサブスタンスＰの測定方法は、加熱処理された尿検体を用いる方法であり、具体的には以下の工程により行うことができる。
［１］尿検体を加熱処理する工程；
［２］［１］で加熱処理された尿検体を用いて、尿検体中のサブスタンスＰを測定する工程；及び、
［３］予め、既知濃度のサブスタンスＰを用いて作成された、サブスタンスＰの濃度と情報量との関係を表す検量線に、［２］での測定で得られた測定値を照らし合わせて、尿検体中のサブスタンスＰの濃度を決定する工程。 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;
　尿検体中のサブスタンスＰの測定は、サブスタンスＰの測定を可能とする方法であればいかなる方法によっても行うことができ、例えば免疫学的測定法等の公知の方法により行うことができる。尿検体中のサブスタンスＰの測定は、市販のキットを用いて行うこともできる。免疫学的測定法は、測定対象成分と、測定対象成分と結合する抗体またはそのフラグメントとの反応（抗原抗体反応）を用いる方法であり、サンドイッチ法、競合法、酵素免疫測定法、化学発光酵素免疫測定法、蛍光免疫測定法、電気化学発光免疫測定法、ラジオイムノアッセイ等が挙げられる。市販のキットとしては、「Substance P EIA Kit」（Cayman Chemical社製）、「Substance P EIA Kit」（Assay Designs社製）、「DRG Human Substance P ELISA (EIA-3120)」（DRGインターナショナル社製）等が挙げられる。 The measurement of substance P in a urine sample can be performed by any method as long as it can measure substance P. For example, 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.
　以下、実施例により本発明を詳細に説明するが、これらは本発明の範囲を何ら限定するものではない。尚、本実施例においては、下記メーカーの試薬を使用した。 Hereinafter, the present invention will be described in detail by way of examples, but these do not limit the scope of the present invention. In this example, reagents from the following manufacturers were used.
（１）コントロール実験
　健常人Ａ，Ｂ，Ｃより採取したそれぞれの新鮮尿（原尿）を、サブスタンスＰ測定用キット「Substance P EIA Kit」（Cayman Chemical社製）中の検体希釈液(EIA Buffer)で希釈することにより、２倍希釈尿、４倍希釈尿、８倍希釈尿を調製した。これらの各尿検体（原尿、２倍希釈尿、４倍希釈尿、８倍希釈尿）中のサブスタンスＰ濃度を、「Substance P EIA Kit」（Cayman Chemical社製）を用いて、当該キットの添付文書に記載された方法に従って決定した。「Substance P EIA Kit」（Cayman Chemical社製）の添付文書には、当該キットを用いる測定の測定範囲として、8.2～500 pg/mLの範囲が記載されている。別途、原尿を４０℃で６０分間加熱処理して得られた尿検体を用いて、同様に、尿検体中のサブスタンスＰ濃度を決定した。測定結果を第１表に示す。第１表中、補正値とは、実際の測定値に希釈倍率を乗じて算出した値である。 (1) Control experiment Each fresh urine (raw urine) collected from healthy persons A, B, and C is diluted with a specimen dilution solution (EIA Buffer) in a substance P measurement kit “Substance P EIA Kit” (Cayman Chemical). 2) diluted urine, 4 times diluted urine and 8 times diluted urine were prepared. The substance P concentration in each of these urine samples (original urine, 2-fold diluted urine, 4-fold diluted urine, 8-fold diluted urine) was determined using the “Substance P EIA Kit” (Cayman Chemical). It was determined according to the method described in the package insert. 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. Separately, 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. In Table 1, the correction value is a value calculated by multiplying the actual measurement value by the dilution factor.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　第１表に示される様に、どの健常人においても、加熱処理を行わない原尿を用いた測定における測定値は、希釈された尿を用いた測定における補正値に比較して、低い値を示した。健常人Ａにおいては、２倍希釈での補正値が、加熱処理した原尿を用いた測定の測定値とほぼ一致した。尚、健常人Ａにおいては、８倍希釈した尿では、希釈された尿中のサブスタンスＰの濃度が非常に低くなり、用いたキットの測定範囲外となったため、正確な測定ができなかった。健常人Ｂにおいては、８倍希釈での補正値が、加熱処理した原尿を用いた測定の測定値とほぼ一致した。健常人Ｃにおいては、８倍希釈での補正値が、加熱処理した原尿を用いた測定の測定値とほぼ一致した。この様に、加熱処理を行わない場合には、正確な測定を行うためには、用いる尿検体によって、希釈倍率を変える必要があるが、本発明の加熱処理を行う方法では、用いる尿検体によらず、単に尿検体を加熱処理するだけでよいことが判明した。従って、本発明の方法により、煩雑な操作を伴うことなく、簡便、かつ、正確に尿検体中のサブスタンスＰを測定できる。 As shown in Table 1, in any healthy person, 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. In the healthy person A, the correction value at the 2-fold dilution almost coincided with the measured value using the heat-treated raw urine. In healthy human A, 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. In the healthy person B, the correction value at the 8-fold dilution almost coincided with the measurement value obtained using the heat-treated raw urine. In the healthy person C, the correction value at the 8-fold dilution almost coincided with the measured value using the heat-treated raw urine. As described above, when heat treatment is not performed, in order to perform accurate measurement, it is necessary to change the dilution factor depending on the urine sample to be used. Regardless, it has been found that it is only necessary to heat-treat the urine sample. Therefore, according to the method of the present invention, the substance P in the urine sample can be measured easily and accurately without complicated operations.
（２）添加回収実験
　加熱処理後の尿検体を用いた測定が、尿検体中のサブスタンスＰの正確な測定であることを、サブスタンスＰの標準品を用いた添加回収実験で検証した。 (2) Addition / recovery experiment It was verified by an addition / recovery experiment using a substance standard of substance P that the measurement using the urine sample after the heat treatment was an accurate measurement of the substance P in the urine sample.
＜添加回収実験＞
１）手順
　健常人より採取した尿、及び、サブスタンスＰ測定用キット「Substance P EIA Kit」（Cayman Chemical社製）中の標準品(1,000 pg/mL)を用いて、以下の様に、添加回収試験用試料(1)～(3)を調製した。試料希釈液は、サブスタンスＰ測定用キット中の希釈液(EIA Buffer)を用いた。
　(1)尿　　　　　９容　＋　試料希釈液　１容
　(2)尿　　　　　９容　＋　標準品　　　１容
　(3)試料希釈液　９容　＋　標準品　　　１容 <Additive recovery experiment>
1) Procedure Using urine collected from a healthy person and a standard product (1,000 pg / mL) in the substance P measurement kit “Substance P EIA Kit” (Cayman Chemical), add and collect as follows: Test samples (1) to (3) were prepared. As the sample diluent, the diluent (EIA Buffer) in the substance P measurement kit was used.
(1) 9 volumes of urine + 1 volume of sample diluent (2) 9 volumes of urine + 1 volume of standard product (3) 9 volumes of sample diluent + 1 volume of standard product
　調製した添加回収試験用試料(1)～(3)それぞれを４０℃で２０～１２０分間、加熱処理し、加熱処理した後の各試料中のサブスタンスＰを、サブスタンスＰ測定用キット「Substance P EIA Kit」（Cayman Chemical社製）を用いて、当該キットの添付文書に記載された方法に従って測定した。 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.
２）データ解析
　添加回収率を下記の式（Ｉ）により算出した。 2) Data analysis The addition recovery rate was calculated by the following formula (I).
Figure JPOXMLDOC01-appb-M000002
Figure JPOXMLDOC01-appb-M000002
　添加回収率の結果を第２表に示す。 The results of the addition recovery rate are shown in Table 2.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　添加回収率が100％に近い程、正確な測定であることを意味する。第２表に示される様に、４０℃で６０分間以上加熱処理された尿検体において、良好な添加回収率を確認した。従って、４０℃で６０分間以上加熱処理された尿検体を用いた測定が、尿検体中のサブスタンスＰの正確な測定であることが判明した。 ¡The closer the addition recovery rate is to 100%, the more accurate the measurement. As shown in Table 2, a good addition recovery rate was confirmed in a urine sample heat-treated at 40 ° C. for 60 minutes or more. Therefore, it has been found that the measurement using the urine sample heat-treated at 40 ° C. for 60 minutes or more is an accurate measurement of the substance P in the urine sample.
・加熱処理条件の検討
　健常人より採取した尿検体（原尿）を、３０℃、３５℃、４０℃、４５℃、５０℃及び５５℃の各温度で加熱した後、当該加熱処理された尿検体を用いて、尿検体中のサブスタンスＰを、サブスタンスＰ測定用キット「Substance P EIA Kit」（Cayman Chemical社製）を用いて、当該キットの添付文書に記載された方法に従って測定した。測定結果を第３表に示す。 -Examination of heat treatment conditions 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.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　ここで、第３表中の数値は、４０℃で６０分間加熱処理された尿検体を用いた測定での測定値を１００とした時の相対値（％）を表す。第３表から、尿検体の加熱温度により、正確な測定が可能となる加熱時間が異なり、加熱温度が高い程、加熱時間が短縮されると共に、長時間の加熱では却って、測定値が低下することが判明した。 Here, 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.
・尿検体の加熱条件の検討
　健常人より採取した尿検体（原尿）を、第４表に示す１０の条件で処理し、当該処理された尿検体中のサブスタンスＰを、サブスタンスＰ測定用キット「Substance P EIA Kit」（Cayman Chemical社製）を用いて、当該キットの添付文書に記載された方法に従って測定した。測定結果を第４表に示す。 -Examination of heating conditions for urine specimens A urine specimen (original urine) collected from a healthy person is treated under the conditions shown in Table 4, and the substance P in the treated urine specimen is treated with a substance P measurement kit. Using “Substance P EIA Kit” (manufactured by Cayman Chemical), measurement was performed according to the method described in the package insert of the kit. The measurement results are shown in Table 4.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　ここで、第４表中の測定値とは、４０℃で６０分間加熱処理された尿検体を用いた測定（条件３）における測定値を１００とした時の相対値（％）を意味する。第４表から、加熱処理された尿検体（条件３～６、９～１０）を用いた場合には、尿検体中のサブスタンスＰを正確に測定できるのに対して、加熱処理されていない尿検体（条件１～２、７～８）を用いた場合には、測定値が著しく低くなり、これらの尿検体では、尿検体中のサブスタンスＰを正確に測定できないことが判明した。本結果から、尿検体を加熱処理することにより、尿検体中のサブスタンスＰを正確に測定できることが判明した。 Here, 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.
　本発明により、炎症やアレルギー疾患の診断に有用な、尿検体中のサブスタンスＰの簡便、かつ、正確な測定方法が提供される。
  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.

Claims (5)

  1. 尿検体を加熱処理し、加熱処理後の尿検体中のサブスタンスＰを測定することを特徴とする、尿検体中のサブスタンスＰの測定方法。 A method for measuring substance P in a urine sample, which comprises subjecting the urine sample to heat treatment and measuring substance P in the urine sample after heat treatment.
  2. 加熱処理が、尿検体を３０～５５℃で加熱する処理である、請求項１記載の測定方法。 The measurement method according to claim 1, wherein the heat treatment is a treatment of heating the urine sample at 30 to 55 ° C.
  3. 加熱処理が、尿検体を３５～５０℃で、１５分間～４時間加熱する処理である、請求項１または２記載の測定方法。 The measurement method according to claim 1 or 2, wherein the heat treatment is a treatment in which the urine sample is heated at 35 to 50 ° C for 15 minutes to 4 hours.
  4. サブスタンスＰの測定が、免疫学的測定法により行われる、請求項１～３のいずれかに記載の測定方法。 The measurement method according to any one of claims 1 to 3, wherein the substance P is measured by an immunological measurement method.
  5. 尿検体が、原尿である、請求項１～４のいずれかに記載の測定方法。
          The measurement method according to claim 1, wherein the urine sample is raw urine.
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