JPH07155198A - Method for measuring pepsiongen in urine - Google Patents
Method for measuring pepsiongen in urineInfo
- Publication number
- JPH07155198A JPH07155198A JP33947793A JP33947793A JPH07155198A JP H07155198 A JPH07155198 A JP H07155198A JP 33947793 A JP33947793 A JP 33947793A JP 33947793 A JP33947793 A JP 33947793A JP H07155198 A JPH07155198 A JP H07155198A
- Authority
- JP
- Japan
- Prior art keywords
- pepsinogen
- urine
- urinary
- enzyme
- pepsin
- 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.)
- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims abstract description 40
- 210000002700 urine Anatomy 0.000 title claims abstract description 31
- 108010047320 Pepsinogen A Proteins 0.000 claims abstract description 60
- 102000004190 Enzymes Human genes 0.000 claims abstract description 48
- 108090000790 Enzymes Proteins 0.000 claims abstract description 48
- 229940088598 enzyme Drugs 0.000 claims abstract description 48
- 102000057297 Pepsin A Human genes 0.000 claims abstract description 39
- 108090000284 Pepsin A Proteins 0.000 claims abstract description 39
- 229940111202 pepsin Drugs 0.000 claims abstract description 39
- 230000000694 effects Effects 0.000 claims abstract description 29
- 238000005259 measurement Methods 0.000 claims abstract description 17
- 230000002378 acidificating effect Effects 0.000 claims abstract description 16
- 230000002485 urinary effect Effects 0.000 claims description 44
- 239000002253 acid Substances 0.000 claims description 32
- 108091005508 Acid proteases Proteins 0.000 claims description 2
- 238000000691 measurement method Methods 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims 1
- 239000000243 solution Substances 0.000 abstract description 24
- 208000005718 Stomach Neoplasms Diseases 0.000 abstract description 7
- 208000007107 Stomach Ulcer Diseases 0.000 abstract description 7
- 206010017758 gastric cancer Diseases 0.000 abstract description 7
- 201000005917 gastric ulcer Diseases 0.000 abstract description 7
- 201000011549 stomach cancer Diseases 0.000 abstract description 7
- 239000007853 buffer solution Substances 0.000 abstract description 6
- 108010079570 uropepsin Proteins 0.000 abstract description 4
- 108091005804 Peptidases Proteins 0.000 abstract description 2
- 239000004365 Protease Substances 0.000 abstract description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 abstract description 2
- 239000004202 carbamide Substances 0.000 abstract description 2
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 239000003153 chemical reaction reagent Substances 0.000 description 15
- 238000012937 correction Methods 0.000 description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 9
- 238000012216 screening Methods 0.000 description 9
- 108090000631 Trypsin Proteins 0.000 description 8
- 102000004142 Trypsin Human genes 0.000 description 8
- 239000012588 trypsin Substances 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 6
- 108090000317 Chymotrypsin Proteins 0.000 description 5
- 229960002376 chymotrypsin Drugs 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 229940122618 Trypsin inhibitor Drugs 0.000 description 3
- 101710162629 Trypsin inhibitor Proteins 0.000 description 3
- 238000011088 calibration curve Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000002753 trypsin inhibitor Substances 0.000 description 3
- 208000007882 Gastritis Diseases 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- 229940109239 creatinine Drugs 0.000 description 2
- DTPCFIHYWYONMD-UHFFFAOYSA-N decaethylene glycol Polymers OCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO DTPCFIHYWYONMD-UHFFFAOYSA-N 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- ZPIRTVJRHUMMOI-UHFFFAOYSA-N octoxybenzene Chemical compound CCCCCCCCOC1=CC=CC=C1 ZPIRTVJRHUMMOI-UHFFFAOYSA-N 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 208000004300 Atrophic Gastritis Diseases 0.000 description 1
- 208000036495 Gastritis atrophic Diseases 0.000 description 1
- DEOKFPFLXFNAON-UHFFFAOYSA-N N-α-Benzoyl-DL-arginine 4-nitroanilide hydrochloride Chemical compound Cl.C=1C=C([N+]([O-])=O)C=CC=1NC(=O)C(CCCN=C(N)N)NC(=O)C1=CC=CC=C1 DEOKFPFLXFNAON-UHFFFAOYSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000016644 chronic atrophic gastritis Diseases 0.000 description 1
- 208000023652 chronic gastritis Diseases 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 208000000718 duodenal ulcer Diseases 0.000 description 1
- YMTINGFKWWXKFG-UHFFFAOYSA-N fenofibrate Chemical compound C1=CC(OC(C)(C)C(=O)OC(C)C)=CC=C1C(=O)C1=CC=C(Cl)C=C1 YMTINGFKWWXKFG-UHFFFAOYSA-N 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002834 transmittance Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、胃潰瘍、胃癌等の診断
に利用される、尿中ペプシノーゲン（ウロペプシン）の
測定方法に関する。TECHNICAL FIELD The present invention relates to a method for measuring urinary pepsinogen (uropepsin), which is used for diagnosing gastric ulcer, gastric cancer and the like.
【０００２】[0002]
【発明の背景】ペプシノーゲンは、酸性条件下でペプシ
ンとなる、ペプシンの不活性型前駆体であり、尿中にも
存在することは古くから知られている。尿中ペプシノー
ゲン（ウロペプシン）は、胃主細胞から分泌されたペプ
シノーゲンが血中に移行した後に腎臓から排泄されたも
ので、その量は胃ペプシン量と関係があり、例えばスト
レス下では鋭敏に増加し、例えば十二指腸潰瘍、胃潰
瘍、急性胃炎等の時には異常高値を示し、例えば萎縮性
胃炎（慢性胃炎）、胃癌等の時には異常低値を示すとい
われている。BACKGROUND OF THE INVENTION Pepsinogen is an inactive precursor of pepsin that turns into pepsin under acidic conditions, and has long been known to exist in urine. Urinary pepsinogen (uropepsin) is excreted from the kidney after pepsinogen secreted from gastric main cells is transferred into the blood, and its amount is related to the amount of gastric pepsin, and increases sharply under stress, for example. It is said that, for example, it exhibits an abnormally high value in the case of duodenal ulcer, gastric ulcer, acute gastritis, etc., and exhibits an abnormally low value in, for example, atrophic gastritis (chronic gastritis), gastric cancer and the like.
【０００３】また、尿は、血液や血清等に比べて採取が
容易であるため、胃潰瘍、胃癌等のスクリーニング（集
団検診）に尿中ペプシノーゲンの測定が利用できればそ
の意義は大きい。Since urine can be collected more easily than blood and serum, it is of great significance if the measurement of urinary pepsinogen can be used for screening for gastric ulcer, gastric cancer and the like (group screening).
【０００４】しかしながら、現在行われている主な尿中
ペプシノーゲンの測定方法である凝乳法［臨床検査法提
要、金井正光編、689頁、昭和60年11月20日発行第29版
第５刷、金原出版(株)等］は、測定操作が煩雑で自動分
析装置に応用し難い方法であり、且つ１検体の測定に約
１時間かかる場合もあるという問題点をも有している方
法であるため、スクリーニング（集団検診）に利用する
のに適したものとは言い難い。[0004] However, the curdling method, which is the main method for measuring urinary pepsinogen currently in use, [Clinical Examination Method Recommendation, edited by Masamitsu Kanai, p. 689, published on November 20, 1985, 29th edition, 5th edition] , Kanahara Publishing Co., Ltd.] is a method that is difficult to apply to an automatic analyzer due to its complicated measurement operation, and it has a problem that it may take about 1 hour to measure one sample. Therefore, it is hard to say that it is suitable for use in screening (group screening).
【０００５】また、最近、放射免疫測定法（ＲＩＡ）に
よる血中ペプシノーゲン測定法が開発されたが、この方
法も測定操作が煩雑であったり、特別な施設を使用する
必要があることや廃棄物処理上の問題があるため、スク
リーニング（集団検診）に利用するのに適したものとは
言い難い。そのため、尿中ペプシノーゲンの簡便で且つ
精度の高い測定法の開発が期待されている現状にある。Recently, a method for measuring blood pepsinogen by a radioimmunoassay (RIA) has been developed. However, this method also requires complicated measuring operations, requires the use of special facilities, and wastes. Due to processing problems, it is difficult to say that it is suitable for use in screening (group screening). Therefore, the development of a simple and highly accurate measurement method for urinary pepsinogen is expected.
【０００６】[0006]
【発明の目的】本発明は上記した如き状況に鑑み成され
たもので、胃潰瘍、胃癌等の診断に利用される、尿中ペ
プシノーゲン（ウロペプシン）を簡便に且つ精度良く測
定する方法を提供することをその目的とする。SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and provides a method for simply and accurately measuring urinary pepsinogen (uropepsin), which is used for the diagnosis of gastric ulcer, gastric cancer and the like. Is its purpose.
【０００７】[0007]
【発明の構成】本発明は、尿中ペプシノーゲンが活性化
される酸性条件下では変性しないが活性化された尿中ペ
プシノーゲン（ペプシン）により加水分解されて失活す
る性質と、活性化された尿中ペプシノーゲンが不活化さ
れるpＨ条件下で活性測定を行うことができる性質とを
有する酵素（以下、耐酸性酵素と略記する。）と尿と
を、尿中ペプシノーゲンが活性化される酸性条件下で反
応させた後、反応液のpＨを尿中ペプシンが不活化され
るpＨに調製して耐酸性酵素の活性測定を行い、得られ
た該酵素活性値に基づいて尿中ペプシノーゲン量を測定
することを特徴とする、尿中ペプシノーゲンの測定方
法、の発明である。The present invention has the property that urinary pepsinogen is not denatured under activated acidic conditions but is hydrolyzed and inactivated by activated urinary pepsinogen (pepsin) and activated urine. An enzyme (hereinafter, abbreviated as an acid-resistant enzyme) having the property that activity can be measured under pH conditions in which middle pepsinogen is inactivated, and urine are treated under acidic conditions in which urinary pepsinogen is activated. After the reaction, the pH of the reaction solution is adjusted to a pH that inactivates urinary pepsin, the activity of acid-resistant enzyme is measured, and the urinary pepsinogen amount is measured based on the obtained enzyme activity value. An invention of a method for measuring urinary pepsinogen, which is characterized in that:
【０００８】即ち、本発明者らは、尿中ペプシノーゲン
を簡便に精度良く且つ高感度に測定し得る方法を見出す
べく鋭意研究の結果、尿中ペプシノーゲンが活性化され
る酸性条件下では変性しないが活性化された尿中ペプシ
ノーゲン（ペプシン）により加水分解されて失活する性
質と、活性化された尿中ペプシノーゲンが不活化される
pＨ条件下で活性測定を行うことができる性質とを有す
る酵素（耐酸性酵素）を尿中ペプシノーゲンが活性化さ
れるpＨ範囲で尿中ペプシノーゲンと反応させてその一
部を失活させた後、一旦活性化された尿中ペプシノーゲ
ン（ペプシン）が不活化されるようなpＨ範囲で該耐酸
性酵素の活性測定を行い、得られた該耐酸性酵素の活性
値を利用することにより尿中ペプシノーゲン量を簡便に
精度良く且つ高感度に測定できることを見出し、本発明
を完成するに至った。[0008] That is, the inventors of the present invention have made earnest studies to find a method capable of simply and accurately measuring urinary pepsinogen with high sensitivity. As a result, the present invention does not denature under acidic conditions in which urinary pepsinogen is activated. Hydrolyzed by activated urinary pepsinogen (pepsin) and inactivated, and activated urinary pepsinogen is inactivated.
After inactivating a part of the enzyme (acid-resistant enzyme) having the property that activity can be measured under pH conditions (acid-resistant enzyme) to react with urinary pepsinogen in the pH range where urinary pepsinogen is activated, The amount of urinary pepsinogen measured by measuring the activity of the acid-resistant enzyme in the pH range where once activated urinary pepsinogen (pepsin) is inactivated, and using the obtained activity value of the acid-resistant enzyme. The inventors have found that it can be easily and accurately measured with high sensitivity, and completed the present invention.
【０００９】本発明の測定方法に於いて使用される耐酸
性酵素としては、尿中ペプシノーゲンが活性化されてペ
プシンとなるpＨ範囲では変性されないが該ペプシンに
より加水分解されて失活する性質と、該ペプシンが不活
化されるようなpＨ範囲で活性測定が可能な性質とを有
する酵素であれば特に限定されることなく挙げられる
が、例えばトリプシン，キモトリプシン等の酸性プロテ
アーゼ等が好ましく挙げられる。これらの使用濃度とし
ては、尿中ペプシノーゲンの活性測定が行える範囲であ
れば特に限定することなく挙げられるが、酸性条件下で
尿と反応させる際の反応液中の濃度として通常0.5〜5.0
mg/dl、好ましくは１〜３mg/dlの範囲が挙げられる。The acid-resistant enzyme used in the assay method of the present invention has the property that it is not denatured in the pH range where urinary pepsinogen is activated to pepsin, but it is hydrolyzed and inactivated by the pepsin. There is no particular limitation as long as it is an enzyme having the property of being able to measure the activity in the pH range such that the pepsin is inactivated. For example, acidic proteases such as trypsin and chymotrypsin are preferable. The concentration of these used is not particularly limited as long as it can measure the activity of urinary pepsinogen, but usually 0.5 to 5.0 as the concentration in the reaction solution when reacting with urine under acidic conditions.
The range is mg / dl, preferably 1 to 3 mg / dl.
【００１０】本発明を実施するには例えば以下の如くし
て行えばよい。即ち、先ず尿中ペプシノーゲンが活性化
されてペプシンとなるpＨ範囲、より具体的には通常pＨ
1.5〜5.0、好ましくはpＨ1.8〜3.0の範囲に調整した緩
衝液（例えばリン酸緩衝液、グリシン・塩酸緩衝液、ク
エン酸緩衝液等）等に、検体尿と耐酸性酵素とを添加し
て例えば25〜37℃で５〜10分間程度反応させた後、反応
液のpＨをペプシンは失活するが耐酸性酵素の活性測定
が行えるpＨ範囲に再度調整して耐酸性酵素の活性測定
を常法により行う。得られた該活性値を、予め濃度既知
のペプシン溶液を検体尿の代りに用い、上記と同じ試薬
を用い、同様の操作を行って得られた、ペプシン量と耐
酸性酵素活性値との関係を表わす検量線に当てはめるこ
とにより検体尿中のペプシノーゲン量を求めることがで
きる。To carry out the present invention, for example, the following may be carried out. That is, first, the urinary pepsinogen is activated to pepsin, and more specifically, the normal pH range.
The sample urine and the acid-resistant enzyme are added to a buffer solution (eg, phosphate buffer solution, glycine / hydrochloric acid buffer solution, citrate buffer solution, etc.) adjusted to a range of 1.5 to 5.0, preferably pH 1.8 to 3.0. For example, after reacting at 25 to 37 ° C for about 5 to 10 minutes, the pH of the reaction solution is adjusted again to a pH range in which the activity of acid-resistant enzyme can be measured although pepsin is inactivated, and the activity of acid-resistant enzyme is measured. It is carried out by a conventional method. The obtained activity value was obtained by performing a similar operation using a pepsin solution of a known concentration in place of the sample urine, using the same reagent as above, and the relationship between the amount of pepsin and the acid-resistant enzyme activity value. The amount of pepsinogen in the urine of the sample can be determined by fitting it to the calibration curve representing
【００１１】上記の測定に於いて、耐酸性酵素として例
えばトリプシンやキモトリプシン等尿中にそのインヒビ
ターが存在する酵素を使用する場合には、注意が必要で
ある。即ち、尿中に存在するトリプシンインヒビターに
よりトリプシン等が失活するため測定値に正誤差が生ず
る場合があるからである。このような測定への影響を避
けるためには、予め検体尿中のトリプシンインヒビター
によるトリプシンの失活量を盲検値として測定し、この
値を上記の操作により得られる耐酸性酵素活性値から差
し引く等の処置を行うことが望ましい。尚、該盲検値を
求める方法としては、トリプシンインヒビターによるト
リプシン等の失活量を測定し得る方法であれば特に限定
されない。In the above measurement, caution is required when using an enzyme such as trypsin or chymotrypsin whose inhibitor is present in urine as an acid resistant enzyme. That is, the trypsin inhibitor present in urine deactivates trypsin and the like, which may cause a positive error in the measured value. In order to avoid such an influence on the measurement, the amount of inactivation of trypsin by the trypsin inhibitor in the urine of the sample is measured as a blind value, and this value is subtracted from the acid-resistant enzyme activity value obtained by the above operation. It is desirable to take measures such as The method for obtaining the blinded value is not particularly limited as long as it is a method capable of measuring the amount of inactivated trypsin or the like by the trypsin inhibitor.
【００１２】本発明を実施する際の、ペプシンは失活す
るが耐酸性酵素の活性測定が行えるpＨ範囲は、耐酸性
酵素の種類により異なるが、例えば耐酸性酵素としてト
リプシン、キモトリプシン等の酸性プロテアーゼを使用
する際には通常pＨ7.2〜9.0、好ましくはpＨ7.6〜8.5の
範囲が挙げられる。In carrying out the present invention, pepsin is inactivated, but the pH range in which the activity of an acid-resistant enzyme can be measured varies depending on the type of the acid-resistant enzyme. For example, an acid protease such as trypsin or chymotrypsin is used as the acid-resistant enzyme. When used, pH is usually in the range of 7.2 to 9.0, preferably pH 7.6 to 8.5.
【００１３】また、耐酸性酵素の活性測定は、該耐酸性
酵素の活性測定の常法に準じて実施すればよく、使用す
る基質、緩衝液、賦活剤等の試薬類も常法に於いて使用
されるものの中から適宜選択して使用すれば足りるし、
これらの使用濃度も常法に於ける使用濃度範囲から適宜
選択して決定すれば足りる。尚、耐酸性酵素の基質は、
酸性条件下で変性されず且つペプシンにより分解されな
い性質を有するものであれば、耐酸性酵素と尿中ペプシ
ノーゲン（ペプシン）との反応時に共存させておいても
よいことは言うまでもない。The activity of the acid-resistant enzyme may be measured in accordance with the usual method for measuring the activity of the acid-resistant enzyme, and the reagents to be used such as the substrate, buffer solution and activator are also used in the usual method. It suffices to use it by appropriately selecting from the used ones.
It suffices that these use concentrations are appropriately selected and determined from the use concentration range in the conventional method. The substrate for the acid-resistant enzyme is
It goes without saying that the acid-resistant enzyme and the urine pepsinogen (pepsin) may be allowed to coexist during the reaction as long as the acid-resistant enzyme is not denatured under acidic conditions and is not decomposed by pepsin.
【００１４】本発明の尿中ペプシノーゲンの測定に於い
ては、ペプシンにより分解されて失活する性質を有する
耐酸性酵素を利用して測定を行うため、極めて短時間で
目的の測定を行うことができる。即ち、耐酸性酵素はペ
プシンにより加水分解されて容易に失活するので反応時
間を短時間とした場合でも、活性化された尿中ペプシノ
ーゲン（ペプシン）量に対応した耐酸性酵素の失活が速
やかに起こるからである。In the measurement of urinary pepsinogen of the present invention, an acid-resistant enzyme having a property of being decomposed by pepsin to be inactivated is used. Therefore, the desired measurement can be performed in an extremely short time. it can. That is, the acid-resistant enzyme is hydrolyzed by pepsin and easily inactivated. Therefore, even when the reaction time is short, the inactivation of the acid-resistant enzyme corresponding to the amount of activated urinary pepsinogen (pepsin) is rapid. Because it happens to.
【００１５】本発明は、上記した如き特徴を有している
ため、自動分析装置を利用した測定への応用が可能であ
るので、胃潰瘍、胃癌等のスクリーニング（集団検診）
用の尿中ペプシノーゲン測定方法として極めて有用な方
法である。以下に参考例、実施例等により、本発明を更
に詳細に述べるが、本発明はこれらにより何等限定され
るものではない。Since the present invention has the characteristics as described above, it can be applied to measurement using an automatic analyzer, and therefore screening for gastric ulcer, gastric cancer, etc. (group medical examination)
It is a very useful method for measuring urinary pepsinogen for urine. Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples, but the present invention is not limited thereto.
【００１６】[0016]
実施例１．尿中ペプシノーゲンの測定 （試料）新鮮尿30検体を試料とした。 （測定試液） ・第１試液 トリプシン（和光純薬工業(株)製、結晶形）を２mg/d
l、N-α-ベンゾイル-DL-アルギニン-p-ニトロアニリド
・塩酸塩（ＢＡＰＮＡ、和光純薬工業(株)製）を60mg/d
l、及びポリオキシエチレン(10)オクチルフェニルエー
テル（和光純薬工業(株)製）を0.1％含む50mMグリシン
・塩酸緩衝液（pＨ2.5）を第１試液とした。 ・第２試液 ポリオキシエチレン(10)オクチルフェニルエーテル（和
光純薬工業(株)製）を0.1％含む１Ｍトリス（ヒドロキ
シメチル）アミノメタン（Tris）・塩酸緩衝液（pＨ8.
0）を第２試液とした。 （ペプシン標準液）ペプシン（結晶ペプシン、シグマ社
製）を所定濃度含む ５mMクエン酸緩衝液（pＨ5.5）を
標準液とした。 （測定機器）日立自動分析装置７０７０型を使用した。 （測定操作）試料25μｌと第１試液200μｌとを混合
し、37℃で５分間インキュベートした後、これに第２試
液200μlを添加、混合した。第２試液添加後１〜３分後
の吸光度変化を測定主波長405nm副波長505nmで測定し、
吸光度変化値１（ΔＥ１）を得た。また、試料25μｌと
第２試液200μｌとを混合し、37℃で５分間インキュベ
ートした後、これに第１試液200μlを添加、混合した。
第１試液添加後１〜３分後の吸光度変化を測定主波長40
5nm副波長505nmで測定し、吸光度変化値２（ΔＥ２）
（盲検値）を得た。得られた２つの吸光度変化値の差
（ΔＥ１−ΔＥ２）を、予め濃度既知のペプシン標準液
を試料として用い、上記と同じ試薬を用い、同様の操作
を行って得られた、ペプシン量と吸光度変化値との関係
を表わす検量線に当てはめることにより、試料中のペプ
シノーゲン量を求めた後、尿中クレアチニン１g当たり
の量（mg/g・CRE）に換算した。 （結果）得られた結果を表１に示す。Example 1. Measurement of urinary pepsinogen (Sample) 30 samples of fresh urine were used as samples. (Measurement reagent) ・ First reagent trypsin (Wako Pure Chemical Industries, Ltd., crystalline form) 2 mg / d
l, N-α-benzoyl-DL-arginine-p-nitroanilide / hydrochloride (BAPNA, manufactured by Wako Pure Chemical Industries, Ltd.) at 60 mg / d
1 and a 50 mM glycine / hydrochloric acid buffer solution (pH 2.5) containing 0.1% of polyoxyethylene (10) octyl phenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the first reagent solution.・ Second reagent solution 1M tris (hydroxymethyl) aminomethane (Tris) containing 0.1% of polyoxyethylene (10) octylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.) ・ hydrochloric acid buffer solution (pH 8.
0) was used as the second reagent solution. (Pepsin standard solution) A 5 mM citrate buffer solution (pH 5.5) containing pepsin (crystal pepsin, manufactured by Sigma) at a predetermined concentration was used as a standard solution. (Measurement equipment) Hitachi automatic analyzer 7070 was used. (Measurement procedure) After mixing 25 μl of the sample and 200 μl of the first reagent solution and incubating at 37 ° C. for 5 minutes, 200 μl of the second reagent solution was added thereto and mixed. The absorbance change 1 to 3 minutes after the addition of the second reagent solution is measured at a main wavelength of 405 nm and a sub wavelength of 505 nm,
The absorbance change value 1 (ΔE1) was obtained. Further, 25 μl of the sample and 200 μl of the second reagent solution were mixed and incubated at 37 ° C. for 5 minutes, and then 200 μl of the first reagent solution was added and mixed.
Measure the change in absorbance 1 to 3 minutes after adding the first reagent solution.
Absorbance change value 2 (ΔE2) measured at 5 nm sub wavelength 505 nm
(Blind value) was obtained. The difference (ΔE1−ΔE2) between the two absorbance change values obtained was obtained by using a pepsin standard solution having a known concentration in advance as a sample, using the same reagent as above, and performing the same operation, and the pepsin amount and the absorbance. The amount of pepsinogen in the sample was determined by applying it to a calibration curve showing the relationship with the change value, and then converted to the amount per 1 g of creatinine in urine (mg / g CRE). (Results) The results obtained are shown in Table 1.
【００１７】参考例１．凝乳法による尿中ペプシノーゲ
ン量の測定 （試料）実施例１と同じものを用いた。 （基質液）市販の牛乳を４Ｍ酢酸緩衝液（pＨ4.5）で18
倍に希釈したものを基質液とした。 （ペプシン標準液）実施例１と同じものを用いた。 （操作法） (1)ペプシン変性試料の調製 試料に６Ｎ塩酸を滴下してpＨを4.0前後に調製した後、
37℃で１時間インキュベートしてペプシン変性試料を得
た。 (2)試料中のペプシノーゲン量の測定 上記(1)で得たペプシン変性試料500μlに、４Ｍ酢酸緩
衝液（pＨ4.5）500μl及び10％尿素水溶液1000μlを添
加、混合した。上記溶液50μlを分注した血小板凝集計
用キュベット（回転子入り）を血小板凝集計（二光バイ
オサイエンス社製、４チャンネルＰＡＴ−４Ａ）にセッ
トし、これに基質液15μlを分注すると同時に４チャン
ネル記録計（二光バイオサイエンス社製、Ｔ−６２６Ｄ
Ｓ）を作動させて記録を開始した。得られたデータをコ
ンピューターで解析し、透過率の最大変化点に到達する
までの時間を求めた。得られた最大変化点到達時間を、
予め濃度既知のペプシン標準液を試料として用い、上記
と同じ試薬を用い、同様の操作を行って得られた、ペプ
シン量と最大変化点到達時間との関係を表わす検量線に
当てはめることにより、試料中のペプシノーゲン量を求
めた後、尿中クレアチニン１g当たりの量（mg/g・CRE）
に換算した。 （結果）得られた結果を表１に併せて示す。Reference Example 1. Measurement of urinary pepsinogen amount by curdling method (Sample) The same sample as in Example 1 was used. (Substrate solution) Commercially available milk is used in 4M acetate buffer (pH 4.5) 18
A one-fold diluted solution was used as a substrate solution. (Pepsin standard solution) The same solution as in Example 1 was used. (Operation method) (1) Preparation of pepsin-denatured sample After 6N hydrochloric acid was added dropwise to the sample to adjust the pH to around 4.0,
Incubation at 37 ° C for 1 hour gave a pepsin-denatured sample. (2) Measurement of the amount of pepsinogen in the sample To 500 μl of the pepsin-denatured sample obtained in (1) above, 500 μl of 4M acetate buffer (pH 4.5) and 1000 μl of 10% urea aqueous solution were added and mixed. The cuvette for platelet aggregometer (with rotor) into which 50 μl of the above solution was dispensed was set in a platelet aggregometer (4 channel PAT-4A manufactured by Nikko Bioscience Co., Ltd.) and 15 μl of the substrate solution was dispensed at the same time. Channel recorder (T-626D, manufactured by Nikko Bioscience)
S) was activated to start recording. The data obtained was analyzed by a computer, and the time required to reach the maximum change point of the transmittance was obtained. The maximum change point arrival time obtained is
Using a pepsin standard solution of known concentration as a sample in advance, using the same reagent as above, and applying the same procedure to the calibration curve representing the relationship between the amount of pepsin and the time to reach the maximum change point, the sample was applied. After determining the amount of pepsinogen in blood, the amount per 1g of creatinine in urine (mg / g ・ CRE)
Converted to. (Results) The obtained results are also shown in Table 1.
【００１８】[0018]
【表１】 ＊相関係数（γ）＝0.9805。 ＊回帰直線式：Ｙ＝1.0973Ｘ−0.1121。 Ｙ：参考例１により得られた値。 Ｘ：実施例１により得られた値。 表１の結果から明らかな如く、本発明の方法は、従来法
である凝乳法と良好な相関を示すことが判る。[Table 1] * Correlation coefficient (γ) = 0.9805. * Regression linear formula: Y = 1.0973X-0.1121. Y: Value obtained in Reference Example 1. X: the value obtained in Example 1. As is clear from the results of Table 1, the method of the present invention shows a good correlation with the conventional coagulation method.
【００１９】[0019]
【発明の効果】以上述べた如く、本発明は、例えば凝乳
法等の従来法に比較して極めて簡便に精度良く且つ高感
度に尿中ペプシノーゲンを測定し得る方法を提供するも
のであり、本発明を利用することにより、自動分析装置
を利用した、胃潰瘍、胃癌等のスクリーニング（集団検
診）用の尿中ペプシノーゲン測定が可能となるので、斯
業に貢献するところ極めて大なる発明である。INDUSTRIAL APPLICABILITY As described above, the present invention provides a method capable of measuring urinary pepsinogen extremely simply, with high precision and with high sensitivity, as compared with conventional methods such as a curd method. By utilizing the present invention, it becomes possible to measure urinary pepsinogen for screening (group screening) for gastric ulcer, gastric cancer and the like using an automatic analyzer, and this is an extremely significant invention that contributes to the industry.
【手続補正書】[Procedure amendment]
【提出日】平成７年１月１８日[Submission date] January 18, 1995
【手続補正１】[Procedure Amendment 1]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】請求項１[Name of item to be corrected] Claim 1
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【手続補正２】[Procedure Amendment 2]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】請求項３[Name of item to be corrected] Claim 3
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【手続補正３】[Procedure 3]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】０００７[Correction target item name] 0007
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【０００７】[0007]
【発明の構成】本発明は、尿中ペプシノーゲンが活性化
される酸性条件下では変性しないが活性化された尿中ペ
プシノーゲン（ペプシン）により加水分解されて失活す
る性質と、活性化された尿中ペプシノーゲンが不活化さ
れるｐＨ条件下で活性測定を行うことができる性質とを
有する酵素（以下、耐酸性酵素と略記する。）と尿と
を、尿中ペプシノーゲンが活性化される酸性条件下で反
応させた後、反応液のｐＨを尿中ペプシンが不活化され
るｐＨに調整して耐酸性酵素の活性測定を行い、得られ
た該酵素活性値に基づいて尿中ペプシノーゲン量を測定
することを特徴とする、尿中ペプシノーゲンの測定方
法、の発明である。The present invention has the property that urinary pepsinogen is not denatured under activated acidic conditions but is hydrolyzed and inactivated by activated urinary pepsinogen (pepsin) and activated urine. An enzyme (hereinafter, abbreviated as acid-resistant enzyme) having the property that activity can be measured under a pH condition in which medium pepsinogen is inactivated and urine, and an acidic condition in which urinary pepsinogen is activated After the reaction, the pH of the reaction solution is adjusted to a pH at which urinary pepsin is inactivated and the activity of the acid-resistant enzyme is measured, and the urinary pepsinogen amount is measured based on the obtained enzyme activity value. An invention of a method for measuring urinary pepsinogen, which is characterized in that:
【手続補正４】[Procedure amendment 4]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】０００９[Correction target item name] 0009
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【０００９】本発明の測定方法に於いて使用される耐酸
性酵素としては、尿中ペプシノーゲンが活性化されてペ
プシンとなるｐＨ範囲では変性されないが該ペプシンに
より加水分解されて失活する性質と、該ペプシンが不活
化されるようなｐＨ範囲で活性測定が可能な性質とを有
する酵素であれば特に限定されることなく挙げられる
が、例えばトリプシン，キモトリプシン等が好ましく挙
げられる。これらの使用濃度としては、尿中ペプシノー
ゲンの活性測定が行える範囲であれば特に限定すること
なく挙げられるが、酸性条件下で尿と反応させる際の反
応液中の濃度として通常０．５〜５．０ｍｇ／ｄｌ、好
ましくは１〜３ｍｇ／ｄｌの範囲が挙げられる。The acid-resistant enzyme used in the measuring method of the present invention has a property that it is not denatured in the pH range where urinary pepsinogen is activated and becomes pepsin, but it is hydrolyzed and inactivated by the pepsin, The enzyme is not particularly limited as long as it is an enzyme having the property that the activity can be measured in the pH range where the pepsin is inactivated. For example, trypsin, chymotrypsin and the like are preferable. There is no particular limitation on the concentration of these used as long as the activity of urinary pepsinogen can be measured, but it is usually 0.5 to 5 as the concentration in the reaction solution when reacted with urine under acidic conditions. The range is 0.0 mg / dl, preferably 1 to 3 mg / dl.
【手続補正５】[Procedure Amendment 5]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】００１２[Correction target item name] 0012
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【００１２】本発明を実施する際の、ペプシンは失活す
るが耐酸性酵素の活性測定が行えるｐＨ範囲は、耐酸性
酵素の種類により異なるが、例えば耐酸性酵素としてト
リプシン、キモトリプシン等を使用する際には通常ｐＨ
７．２〜９．０、好ましくはｐＨ７．６〜５の範囲が挙
げられる。In carrying out the present invention, the pH range in which pepsin is inactivated but the activity of the acid-resistant enzyme can be measured varies depending on the type of the acid-resistant enzyme .
When using lipsin, chymotrypsin, etc., usually pH
The range of 7.2-9.0, preferably pH 7.6-5 is mentioned.
Claims (3)
件下では変性しないが活性化された尿中ペプシノーゲン
（ペプシン）により加水分解されて失活する性質と、活
性化された尿中ペプシノーゲンが不活化されるpＨ条件
下で活性測定を行うことができる性質とを有する酵素
（以下、耐酸性酵素と略記する。）と尿とを、尿中ペプ
シノーゲンが活性化される酸性条件下で反応させた後、
反応液のpＨを尿中ペプシンが不活化されるpＨに調製し
て耐酸性酵素の活性測定を行い、得られた該酵素活性値
に基づいて尿中ペプシノーゲン量を測定することを特徴
とする、尿中ペプシノーゲンの測定方法。1. A property that urinary pepsinogen is not denatured under acidic conditions where it is activated but is hydrolyzed and inactivated by activated urinary pepsinogen (pepsin), and activated urinary pepsinogen is not An enzyme (hereinafter abbreviated as an acid-resistant enzyme) having the property of being able to perform activity measurement under pH condition for activation and urine were reacted under an acidic condition under which urinary pepsinogen was activated. rear,
The pH of the reaction solution is adjusted to a pH that inactivates pepsin in urine, the activity of an acid-resistant enzyme is measured, and the amount of urinary pepsinogen is measured based on the obtained enzyme activity value. Method for measuring urinary pepsinogen.
に記載の測定方法。2. The acidic condition is pH 1.5 to 5.0.
Measurement method described in.
求項１又は２に記載の測定方法。3. The measuring method according to claim 1, wherein the acid-resistant enzyme is an acid protease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33947793A JPH07155198A (en) | 1993-12-03 | 1993-12-03 | Method for measuring pepsiongen in urine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33947793A JPH07155198A (en) | 1993-12-03 | 1993-12-03 | Method for measuring pepsiongen in urine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07155198A true JPH07155198A (en) | 1995-06-20 |
Family
ID=18327844
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP33947793A Withdrawn JPH07155198A (en) | 1993-12-03 | 1993-12-03 | Method for measuring pepsiongen in urine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07155198A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5856117A (en) * | 1996-06-21 | 1999-01-05 | Kyoto Daiichi Kagaku Co., Ltd. | Method for measuring the concentration of protease inhibitors, kit for use in such a method and method for dissolving a substrate |
| US6130055A (en) * | 1997-08-29 | 2000-10-10 | Kyoto Daiichi Kagaku Co., Ltd. | Method for measuring the concentration or the activity of protease inhibitor |
-
1993
- 1993-12-03 JP JP33947793A patent/JPH07155198A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5856117A (en) * | 1996-06-21 | 1999-01-05 | Kyoto Daiichi Kagaku Co., Ltd. | Method for measuring the concentration of protease inhibitors, kit for use in such a method and method for dissolving a substrate |
| US6130055A (en) * | 1997-08-29 | 2000-10-10 | Kyoto Daiichi Kagaku Co., Ltd. | Method for measuring the concentration or the activity of protease inhibitor |
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