JPH0313880B2 - - Google Patents
Info
- Publication number
- JPH0313880B2 JPH0313880B2 JP60236081A JP23608185A JPH0313880B2 JP H0313880 B2 JPH0313880 B2 JP H0313880B2 JP 60236081 A JP60236081 A JP 60236081A JP 23608185 A JP23608185 A JP 23608185A JP H0313880 B2 JPH0313880 B2 JP H0313880B2
- Authority
- JP
- Japan
- Prior art keywords
- reagent
- acid phosphatase
- nitrophenol
- present
- phosphatase activity
- 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.)
- Expired - Lifetime
Links
- 239000003153 chemical reaction reagent Substances 0.000 claims description 31
- 230000000694 effects Effects 0.000 claims description 20
- 239000000758 substrate Substances 0.000 claims description 13
- 125000005843 halogen group Chemical group 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 5
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims 1
- 230000002378 acidificating effect Effects 0.000 claims 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 28
- 108010051457 Acid Phosphatase Proteins 0.000 description 28
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 27
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 238000005259 measurement Methods 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 6
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000007979 citrate buffer Substances 0.000 description 5
- 239000012085 test solution Substances 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 210000001124 body fluid Anatomy 0.000 description 4
- 239000010839 body fluid Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- -1 2,3,6-trichloro-4-nitrophenyl phosphate Chemical compound 0.000 description 3
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 3
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 238000004040 coloring Methods 0.000 description 3
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- OBFPUWKJCNIVNM-UHFFFAOYSA-N (2,6-dichloro-4-nitrophenyl) dihydrogen phosphate Chemical compound OP(O)(=O)OC1=C(Cl)C=C([N+]([O-])=O)C=C1Cl OBFPUWKJCNIVNM-UHFFFAOYSA-N 0.000 description 2
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- IKQSNKKSCOCCOD-UHFFFAOYSA-N molport-028-600-099 Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1Cl IKQSNKKSCOCCOD-UHFFFAOYSA-N 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 2
- XNGIFLGASWRNHJ-UHFFFAOYSA-N phthalic acid Chemical compound OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OSAJZHUCWQMEDB-UHFFFAOYSA-N 2,3,6-tribromo-4-nitrophenol Chemical compound OC1=C(Br)C=C([N+]([O-])=O)C(Br)=C1Br OSAJZHUCWQMEDB-UHFFFAOYSA-N 0.000 description 1
- UAXQXSVCGHEVMI-UHFFFAOYSA-N 2,3,6-trichloro-4-nitrophenol Chemical compound OC1=C(Cl)C=C([N+]([O-])=O)C(Cl)=C1Cl UAXQXSVCGHEVMI-UHFFFAOYSA-N 0.000 description 1
- YCDOXMDERHDBDV-UHFFFAOYSA-N 2,3,6-triiodo-4-nitrophenol Chemical compound OC1=C(I)C=C([N+]([O-])=O)C(I)=C1I YCDOXMDERHDBDV-UHFFFAOYSA-N 0.000 description 1
- PXSGFTWBZNPNIC-UHFFFAOYSA-N 618-80-4 Chemical compound OC1=C(Cl)C=C([N+]([O-])=O)C=C1Cl PXSGFTWBZNPNIC-UHFFFAOYSA-N 0.000 description 1
- BOFRXDMCQRTGII-UHFFFAOYSA-N 619-08-9 Chemical compound OC1=CC=C([N+]([O-])=O)C=C1Cl BOFRXDMCQRTGII-UHFFFAOYSA-N 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- UVGTXNPVQOQFQW-UHFFFAOYSA-N Disophenol Chemical compound OC1=C(I)C=C([N+]([O-])=O)C=C1I UVGTXNPVQOQFQW-UHFFFAOYSA-N 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- MQRJBSHKWOFOGF-UHFFFAOYSA-L disodium;carbonate;hydrate Chemical compound O.[Na+].[Na+].[O-]C([O-])=O MQRJBSHKWOFOGF-UHFFFAOYSA-L 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- RBXVOQPAMPBADW-UHFFFAOYSA-N nitrous acid;phenol Chemical class ON=O.OC1=CC=CC=C1 RBXVOQPAMPBADW-UHFFFAOYSA-N 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 150000003014 phosphoric acid esters Chemical class 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- RPACBEVZENYWOL-XFULWGLBSA-M sodium;(2r)-2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate Chemical compound [Na+].C=1C=C(Cl)C=CC=1OCCCCCC[C@]1(C(=O)[O-])CO1 RPACBEVZENYWOL-XFULWGLBSA-M 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
(産業上の利用分野)
本発明は酸性フオスフアターゼ活性測定用試薬
に関するものである。体液中の酸性フオスフアタ
ーゼ活性の測定は、前立腺癌の診断及び経過観察
に有用な情報を与えるものとして臨床意義が高
い。
(従来の技術)
従来、酸性フオスフアターゼ活性はリン酸の水
酸基にp−ニトロフエノールを結合させた基質を
用いて酸性フオスフアターゼを作用させ、遊離し
てくるp−ニトロフエノールをアルカリ性下で比
色する方法や酵素イムノアツセイ(以下EIAと略
す)、ラジオイムノアツセイ(以下RIAと略す)
による方法が用いられていた。
ところがp−ニトロフエノールを用いた方法で
は目的とする酵素酸性フオスフアターゼの至適PH
(PH5.0付近)と発色基であるp−ニトロフエノー
ルの発色PH(PH9以上)とが異なる為に酸性フオ
スフアターゼ活性を測定する為には酵素反応と発
色対応を別々に行なう必要がある。その為に試薬
数及び操作ステツプが多く必要となり、酵素活性
を求める場合に一番適当であるといわれている速
度分析(レートアツセイ)法が出来ない欠点があ
る。
EIA、RIAによる方法では、測定が長時間を有
し操作の煩雑さ等に欠点がある。
(発明の解決しようとする問題点)
本発明の目的は定量性に優れた酸性フオスフア
ターゼのレートアツセイが可能となる酸性フオス
フアターゼ活性測定試薬を提供することである。
(問題点を解決する為の手段)
本発明者らは、上記目的を達成するために種々
鋭意検討したところ、一般式〔〕で示される基
質を用いることにより体液中の酸性フオスフアタ
ーゼ活性を短時間に正確簡単にレートアツセイ出
来ることを見い出し本発明に到達した。
すなわち、本発明は基質として下記一般式
〔〕で示される化合物を含有することを特徴と
する酸性フオスフアターゼ活性測定試薬である。
(式中、Xはハロゲン原子を示し、かつYは水
素原子またはハロゲン原子を示す。)
本発明に用いる基質としては一般式〔〕で示
される化合物、すなわちリン酸の1つの水産基が
少なくとも2個のハロゲン原子が2,6−位に置
換し、1個のニトロ基が4−位に置換したフエニ
ル基と結合したものである。少なくとも2個のハ
ロゲン原子が2,6一位に置換し、1個のニトロ
基が4−位に置換したフエニル基としては、解裂
したアグリコンが基質と異なつた吸収スペクトル
を示すものであり、かつpkaが低く酸性フオスフ
アターゼの測定域PH5.0付近で充分に発色解離す
る化合物を生ずるものである。
解裂したアグリコンとは具体的には次の一般式:
(X、Yは前記のものと同じ)で示されるフエ
ノール誘導体である。
例えば、2,6−ジクロロ−4−ニトロフエノ
ール、2,6−ジプロモ−4−ニトロフエノー
ル、2,6−ジヨード−4−ニトロフエノール、
2,3,6−トリクロロ−4−ニトロフエノー
ル、2,3,6−トリブロモ−4−ニトロフエノ
ール、2,3,6−トリヨード−4−ニトロフエ
ノール等があげられる。
これら基質の合成方法は、例えばハロゲン置換
のp−ニトロフエノールにフオスフオリルクロラ
イドを反応させてリン酸エステル化し
(Chemical Abstract47 8032b)、さらに脱ハロ
ゲン化し(Bull.Chem.Soc.Japan44 2743)、次
にNa塩とし再結晶化して(J.Biol.Chem.167
57)、目的基質を得る方法がある。
本発明の試薬のPHは体液中の酸性フオスフアタ
ーゼの至適PHである。PH4.0〜5.5を保つ緩衝液で
あれば、いかなるものでも良い。例えばクエン酸
緩衝液やその他有機酸緩衝液、例えば酢酸、コハ
ク酸、フタル酸等の緩衝液があげられる。
基質濃度としては特に制限がないが、好ましく
は最大の酸性フオスフアターゼの酵素活性を示す
濃度が適当である。例えば1mM以上である。
本発明の試薬には必要により、界面活性剤、防
腐剤、塩化ナトリウム、シクロデキストリン、安
定化剤等を加えてもよい。
本発明の酸性フオスフアターゼ活性測定試薬を
用いて酸性フオスフアターゼ活性を測定する方法
としては、試薬を該試薬と反応させて生成するア
グリコンの吸光度の変化を直接分光光度計を用い
て比色定量する方法がある。
(発明の効果)
本発明の酸性フオスフアターゼ活性測定試薬に
おいて、一般式〔〕で示される化合物を基質と
して用いることにより、体液中の酸性フオスフア
ターゼ活性を短時間に正確、かつ簡単にレートア
ツセイすることができる。特にニトロフエノール
を結合した基質に比べて酵素反応と発色反応を1
つの系で行なえるという優れた効果を有する。
また2−クロロ−4−ニトロフエノールなどの
モノハロゲン化ニトロフエノールを結合した基質
に比べて酸性フオスフアターゼの測定域pH5.0付
近で充分に発色解離し、より定量性に優れる。
(実施例)
以下、本発明を実施例により詳細に説明する。
実施例1および比較例1
被検液中の酸性フオスフアターゼ活性量を下記
試薬を用いて下記方法により測定した。
1 試薬A:2−クロロ−4−ニトロフエニルリ
ン酸 1.0mM
クエン酸緩衝液 0.1M(pH4.5)
試薬B:2,3,6−トリクロロ−4−ニトロ
フエニルリン酸 1.0mM
クエン酸緩衝液 0.1M(pH4.5)
試薬C:2,6−ジクロロ−4−ニトロフエニ
ルリン酸 1.0mM
クエン酸緩衝液 0.1M(pH4.5)
2 測定方法
酸性フオスフアターゼ含有被検液50μに上
記試薬A2mlを加えて37℃で反応させ、その吸
光度を400nmにて測定して発色速度を求めた。
反応曲線を第1図に示し、検量線を第2図に示
す。試薬Aに代えて、試薬B又はCを用いて同
様に発色速度を求めた。
第1図および第2図から明らかなように、
2,3,6−トリクロロ−4−ニトロフエニル
リン酸(試薬B)、2,6−ジクロロ−4−ニ
トリロフエニルリン酸(試薬C)を用いた本発
明の試薬では、短時間に正確かつレートアツセ
イすることができ、その感度は2−クロロ−4
−ニトロフエニルリン酸(試薬A)を用いた場
合よりも非常に高い。
実施例2および比較例2
被検液中に酸性フオスフアターゼ活性量を下記
試薬を用いて下記方法により測定した。
1 試薬A:2,3,6−トリクロロ−4−ニト
ロフエニルリン酸 1.0mM
クエン酸緩衝液 0.1M(pH4.5)
試薬B:2,6−ジクロロ−4−ニトロフエニ
ルリン酸 1.0mM
クエン酸緩衝液 0.1M(pH4.5)
試薬C:2−クロロ−4−ニトロフエニルリン
酸 1.0mM
クエン酸緩衝液 0.1M(pH4.5)
試薬D:4−ニトロフエニルリン酸 1.0mM
クエン酸緩衝液 0.1M(pH4.5)
2 測定方法
a 酸性フオスフアターゼ含有被検液(濃度
150U/l)50μに上記試薬A,B,C,又
はD2mlを加えて37℃で3分間加温後、吸光
度変化を波長400nmで測定して1分間の吸
光度変化を求めた(0D/分)。(ブランクは
酸性フオスフアターゼ含有被検液にかわり水
を用いる)
第1表にその結果を示す。
(Industrial Application Field) The present invention relates to a reagent for measuring acid phosphatase activity. Measurement of acid phosphatase activity in body fluids has great clinical significance as it provides useful information for the diagnosis and follow-up of prostate cancer. (Prior art) Conventionally, acid phosphatase activity was determined by using a substrate in which p-nitrophenol was bound to the hydroxyl group of phosphoric acid, allowing acid phosphatase to act, and then colorimetrically measuring the liberated p-nitrophenol under alkaline conditions. , enzyme immunoassay (hereinafter abbreviated as EIA), radioimmunoassay (hereinafter abbreviated as RIA)
The method was used. However, in the method using p-nitrophenol, the optimum pH of the target enzyme acid phosphatase is
(pH around 5.0) and the coloring pH of p-nitrophenol (pH 9 or higher), which is a coloring group, are different. Therefore, in order to measure acid phosphatase activity, it is necessary to perform the enzymatic reaction and coloring separately. Therefore, it requires a large number of reagents and many operational steps, and has the disadvantage that rate assay method, which is said to be the most suitable method for determining enzyme activity, cannot be used. The EIA and RIA methods have drawbacks such as long measurement times and complicated operations. (Problems to be Solved by the Invention) An object of the present invention is to provide a reagent for measuring acid phosphatase activity that enables rate assay of acid phosphatase with excellent quantitative properties. (Means for Solving the Problems) In order to achieve the above object, the present inventors have conducted various intensive studies and found that by using a substrate represented by the general formula [], acid phosphatase activity in body fluids can be suppressed for a short time. We have discovered that it is possible to accurately and easily perform rate assays and have arrived at the present invention. That is, the present invention is a reagent for measuring acid phosphatase activity, which is characterized by containing a compound represented by the following general formula [] as a substrate. (In the formula, X represents a halogen atom, and Y represents a hydrogen atom or a halogen atom.) The substrate used in the present invention is a compound represented by the general formula [ ], that is, one hydroxyl group of phosphoric acid has at least 2 halogen atoms are substituted at the 2,6-position, and one nitro group is bonded to a phenyl group substituted at the 4-position. As a phenyl group in which at least two halogen atoms are substituted at the 2- and 6-1 positions and one nitro group is substituted at the 4-position, the cleaved aglycone exhibits an absorption spectrum different from that of the substrate, Moreover, it produces a compound that has a low pka and is sufficiently colored and dissociated in the measurement range of acid phosphatase, around pH 5.0. Specifically, the cleaved aglycone has the following general formula: It is a phenol derivative represented by (X and Y are the same as above). For example, 2,6-dichloro-4-nitrophenol, 2,6-dipromo-4-nitrophenol, 2,6-diiodo-4-nitrophenol,
Examples thereof include 2,3,6-trichloro-4-nitrophenol, 2,3,6-tribromo-4-nitrophenol, and 2,3,6-triiodo-4-nitrophenol. The method for synthesizing these substrates is, for example, by reacting halogen-substituted p-nitrophenol with phosphoryl chloride to form a phosphoric acid ester (Chemical Abstract 47 8032b), and then dehalogenating it (Bull.Chem.Soc.Japan 44 2743). , and then recrystallized as Na salt (J.Biol.Chem. 167
57), there is a method to obtain the target substrate. The pH of the reagent of the present invention is the optimum pH of acid phosphatase in body fluids. Any buffer solution may be used as long as it maintains a pH of 4.0 to 5.5. Examples include citric acid buffers and other organic acid buffers, such as acetic acid, succinic acid, and phthalic acid buffers. Although there are no particular limitations on the substrate concentration, it is preferably a concentration that exhibits the maximum enzyme activity of acid phosphatase. For example, it is 1 mM or more. If necessary, a surfactant, preservative, sodium chloride, cyclodextrin, stabilizer, etc. may be added to the reagent of the present invention. A method for measuring acid phosphatase activity using the reagent for measuring acid phosphatase activity of the present invention is to directly colorimetrically quantify the change in absorbance of the aglycone produced by reacting the reagent with the reagent using a spectrophotometer. be. (Effect of the invention) In the acid phosphatase activity measurement reagent of the present invention, by using the compound represented by the general formula [ ] as a substrate, acid phosphatase activity in body fluids can be accurately and easily rate assayed in a short time. . In particular, the enzymatic reaction and color reaction are reduced by 1% compared to substrates bound with nitrophenol.
It has the excellent effect of being able to be performed in one system. Furthermore, compared to a substrate bound to a monohalogenated nitrophenol such as 2-chloro-4-nitrophenol, it fully develops and dissociates in the measurement range of acid phosphatase, around pH 5.0, and has better quantitative performance. (Example) Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 and Comparative Example 1 The amount of acid phosphatase activity in the test liquid was measured using the following reagent and the following method. 1 Reagent A: 2-chloro-4-nitrophenyl phosphate 1.0mM citric acid buffer 0.1M (pH 4.5) Reagent B: 2,3,6-trichloro-4-nitrophenyl phosphate 1.0mM citric acid Buffer 0.1M (pH4.5) Reagent C: 2,6-dichloro-4-nitrophenyl phosphate 1.0mM Citrate buffer 0.1M (pH4.5) 2 Measurement method Add the above to 50μ of the acid phosphatase-containing test solution 2 ml of reagent A was added and reacted at 37°C, and the absorbance was measured at 400 nm to determine the color development rate.
The reaction curve is shown in FIG. 1, and the calibration curve is shown in FIG. The color development rate was similarly determined using Reagent B or C instead of Reagent A. As is clear from Figures 1 and 2,
The reagents of the present invention using 2,3,6-trichloro-4-nitrophenyl phosphate (Reagent B) and 2,6-dichloro-4-nitrilophenyl phosphate (Reagent C) can be used accurately in a short time. and rate assay, the sensitivity of which is 2-chloro-4
- much higher than with nitrophenyl phosphate (reagent A). Example 2 and Comparative Example 2 The amount of acid phosphatase activity in the test liquid was measured using the following reagent and the following method. 1 Reagent A: 2,3,6-trichloro-4-nitrophenyl phosphate 1.0mM Citrate buffer 0.1M (pH 4.5) Reagent B: 2,6-dichloro-4-nitrophenyl phosphate 1.0mM Citrate buffer 0.1M (pH4.5) Reagent C: 2-chloro-4-nitrophenyl phosphate 1.0mM Citrate buffer 0.1M (pH4.5) Reagent D: 4-nitrophenyl phosphate 1.0mM Citrate buffer 0.1M (pH4.5) 2 Measurement method a Test solution containing acid phosphatase (concentration
After adding 2 ml of the above reagents A, B, C, or D to 50μ (150 U/l) and heating at 37°C for 3 minutes, the change in absorbance was measured at a wavelength of 400 nm to determine the change in absorbance over 1 minute (0 D/min). . (For the blank, water was used instead of the acid phosphatase-containing test solution.) Table 1 shows the results.
【表】
b 酸性フオスフアターゼ含有被検液(濃度
150U/l)50μに上記試薬A,B,C,又
はD2mlを加え37℃で5分間反応後、0.1M炭
酸ソーダ水を添加し、アルカリ条件下にして
反応を停止させ、波長400nmの吸光度を測
定した(0D)。(ブランクは酸性フオスフア
ターゼ含有被検液にかわり水を用いる)第2
表にその結果を示す。[Table] b Test solution containing acid phosphatase (concentration
Add 2 ml of the above reagents A, B, C, or D to 50μ of 150U/l) and react at 37°C for 5 minutes, then add 0.1M sodium carbonate water to stop the reaction under alkaline conditions, and adjust the absorbance at a wavelength of 400nm. Measured (0D). (For the blank, use water instead of the acid phosphatase-containing test solution.) Second
The results are shown in the table.
【表】
第1表から明らかになるように、本発明の試薬
AおよびBはPH4.5において充分測定可能な感度
を有する試薬であるが、試薬Cは試薬AおよびB
に比較して感度は低い。第1表および第2表から
明らかなように試薬DはPH4.5において全く測定
可能なレベルになく、アルカリ条件においてのみ
測定可能である。
参考例 1
種々のニトロフエノールについて、pKaおよび
酸性フオスフアターゼ活性測定に好適であるPH範
囲、PH5.0でのモル吸光度を測定した。その結果
を第3表に示す。[Table] As is clear from Table 1, reagents A and B of the present invention have sufficient sensitivity for measurement at PH4.5;
Sensitivity is low compared to . As is clear from Tables 1 and 2, Reagent D is not at a measurable level at all at PH4.5 and is only measurable under alkaline conditions. Reference Example 1 For various nitrophenols, the molar absorbance at PH5.0, a pH range suitable for measuring pKa and acid phosphatase activity, was measured. The results are shown in Table 3.
【表】
上記結果から明らかなように、本願発明に使用
するハロニトロフエニルリン酸から解離するニト
ロフエノールはpKaが低く、かつ酸性フオスフア
ターゼ活性測定に好適なPH5.0付近でのモル吸光
係数が高い。したがつて、本願発明に使用する基
質は酸性フオスフアターゼの測定域、PH5.0付近
で充分に発色解離し、測定感度に優れる。[Table] As is clear from the above results, the nitrophenol dissociated from the halonitrophenyl phosphate used in the present invention has a low pKa and a molar extinction coefficient near pH 5.0, which is suitable for measuring acid phosphatase activity. expensive. Therefore, the substrate used in the present invention fully develops and dissociates in the measurement range of acid phosphatase, around pH 5.0, and has excellent measurement sensitivity.
第1図は本発明実施例1及び比較例1の反応曲
線を示す。第2図は本発明実施例1及び比較例1
の検量線を示す。
FIG. 1 shows the reaction curves of Example 1 of the present invention and Comparative Example 1. Figure 2 shows Example 1 of the present invention and Comparative Example 1.
The calibration curve is shown.
Claims (1)
物を含有することを特徴とする酸性フオスフアタ
ーゼ活性測定試薬。 (式中、Xはハロゲン原子を示し、かつYは水素
原子またはハロゲン原子を示す。)[Scope of Claims] 1. A reagent for measuring acidic phosphatase activity, which contains a compound represented by the following general formula [] as a substrate. (In the formula, X represents a halogen atom, and Y represents a hydrogen atom or a halogen atom.)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP23608185A JPS6296099A (en) | 1985-10-22 | 1985-10-22 | Reagent for determination of acidic phosphatase activity |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP23608185A JPS6296099A (en) | 1985-10-22 | 1985-10-22 | Reagent for determination of acidic phosphatase activity |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS6296099A JPS6296099A (en) | 1987-05-02 |
JPH0313880B2 true JPH0313880B2 (en) | 1991-02-25 |
Family
ID=16995439
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP23608185A Granted JPS6296099A (en) | 1985-10-22 | 1985-10-22 | Reagent for determination of acidic phosphatase activity |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6296099A (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2678609B2 (en) * | 1988-02-01 | 1997-11-17 | 和光純薬工業株式会社 | Novel 2-chloronitrophenyl phosphate and method for measuring phosphatase activity using the same |
JP2797102B2 (en) * | 1988-09-20 | 1998-09-17 | 和光純薬工業株式会社 | Acid phosphatase activity reagent |
JP2632391B2 (en) * | 1988-11-16 | 1997-07-23 | 和光純薬工業株式会社 | Method for stabilizing peroxidase |
JP2540717Y2 (en) * | 1991-02-26 | 1997-07-09 | 株式会社大館製作所 | Signal box |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS586480A (en) * | 1981-07-03 | 1983-01-14 | Ricoh Co Ltd | Reflection detector |
-
1985
- 1985-10-22 JP JP23608185A patent/JPS6296099A/en active Granted
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS586480A (en) * | 1981-07-03 | 1983-01-14 | Ricoh Co Ltd | Reflection detector |
Also Published As
Publication number | Publication date |
---|---|
JPS6296099A (en) | 1987-05-02 |
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