JP5334691B2 - Peroxidase chemiluminescence reagent - Google Patents
Peroxidase chemiluminescence reagent Download PDFInfo
- Publication number
- JP5334691B2 JP5334691B2 JP2009134576A JP2009134576A JP5334691B2 JP 5334691 B2 JP5334691 B2 JP 5334691B2 JP 2009134576 A JP2009134576 A JP 2009134576A JP 2009134576 A JP2009134576 A JP 2009134576A JP 5334691 B2 JP5334691 B2 JP 5334691B2
- Authority
- JP
- Japan
- Prior art keywords
- concentration
- pod
- peroxidase
- chemiluminescence
- range
- 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 - Fee Related
Links
- 102000003992 Peroxidases Human genes 0.000 title claims description 32
- 108040007629 peroxidase activity proteins Proteins 0.000 title claims description 32
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 26
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 44
- 238000004020 luminiscence type Methods 0.000 claims description 33
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims description 26
- 239000000126 substance Substances 0.000 claims description 23
- 241000222511 Coprinus Species 0.000 claims description 20
- 239000007800 oxidant agent Substances 0.000 claims description 20
- 239000007853 buffer solution Substances 0.000 claims description 18
- 238000005259 measurement Methods 0.000 claims description 18
- 241000221198 Basidiomycota Species 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 description 55
- 239000000243 solution Substances 0.000 description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 24
- 240000003291 Armoracia rusticana Species 0.000 description 19
- 235000011330 Armoracia rusticana Nutrition 0.000 description 19
- 238000000034 method Methods 0.000 description 18
- 102000004190 Enzymes Human genes 0.000 description 17
- 108090000790 Enzymes Proteins 0.000 description 17
- 239000000872 buffer Substances 0.000 description 16
- 244000005700 microbiome Species 0.000 description 12
- 230000002688 persistence Effects 0.000 description 12
- 239000011780 sodium chloride Substances 0.000 description 12
- 230000009257 reactivity Effects 0.000 description 9
- -1 for example Proteins 0.000 description 8
- 238000003018 immunoassay Methods 0.000 description 8
- 239000012064 sodium phosphate buffer Substances 0.000 description 8
- 238000002372 labelling Methods 0.000 description 7
- 230000001590 oxidative effect Effects 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
- 108010044467 Isoenzymes Proteins 0.000 description 5
- 229920001213 Polysorbate 20 Polymers 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 238000004132 cross linking Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 4
- 239000002184 metal Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 241000222211 Arthromyces Species 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 238000000691 measurement method Methods 0.000 description 3
- 229960003151 mercaptamine Drugs 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241001236797 Coprinellus micaceus Species 0.000 description 2
- 244000251987 Coprinus macrorhizus Species 0.000 description 2
- 235000001673 Coprinus macrorhizus Nutrition 0.000 description 2
- 235000016438 Coprinus micaceus Nutrition 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 2
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 2
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 2
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 102100031013 Transgelin Human genes 0.000 description 2
- ZTOJFFHGPLIVKC-CLFAGFIQSA-N abts Chemical compound S/1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C\1=N\N=C1/SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-CLFAGFIQSA-N 0.000 description 2
- 229910052783 alkali metal Inorganic materials 0.000 description 2
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000011067 equilibration Methods 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- WQYVRQLZKVEZGA-UHFFFAOYSA-N hypochlorite Chemical compound Cl[O-] WQYVRQLZKVEZGA-UHFFFAOYSA-N 0.000 description 2
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 159000000000 sodium salts Chemical class 0.000 description 2
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 2
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 1
- KGLPWQKSKUVKMJ-UHFFFAOYSA-N 2,3-dihydrophthalazine-1,4-dione Chemical class C1=CC=C2C(=O)NNC(=O)C2=C1 KGLPWQKSKUVKMJ-UHFFFAOYSA-N 0.000 description 1
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 1
- HUDPLKWXRLNSPC-UHFFFAOYSA-N 4-aminophthalhydrazide Chemical compound O=C1NNC(=O)C=2C1=CC(N)=CC=2 HUDPLKWXRLNSPC-UHFFFAOYSA-N 0.000 description 1
- VSMDINRNYYEDRN-UHFFFAOYSA-N 4-iodophenol Chemical compound OC1=CC=C(I)C=C1 VSMDINRNYYEDRN-UHFFFAOYSA-N 0.000 description 1
- LEOJISUPFSWNMA-UHFFFAOYSA-N ABEI Chemical compound O=C1NNC(=O)C=2C1=CC(N(CCCCN)CC)=CC=2 LEOJISUPFSWNMA-UHFFFAOYSA-N 0.000 description 1
- 241000222485 Agaricales Species 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000221452 Auriculariales Species 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 241001236839 Coprinellus disseminatus Species 0.000 description 1
- 241001236838 Coprinellus domesticus Species 0.000 description 1
- 241000222432 Coprinopsis radiata Species 0.000 description 1
- 241001236770 Coprinopsis sclerotiger Species 0.000 description 1
- 244000234623 Coprinus comatus Species 0.000 description 1
- 235000004439 Coprinus comatus Nutrition 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 239000006173 Good's buffer Substances 0.000 description 1
- 241000221535 Pucciniales Species 0.000 description 1
- 241000529895 Stercorarius Species 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 229920004896 Triton X-405 Polymers 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000012948 isocyanate Substances 0.000 description 1
- 150000002513 isocyanates Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000036963 noncompetitive effect Effects 0.000 description 1
- 150000001451 organic peroxides Chemical class 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- CZPZWMPYEINMCF-UHFFFAOYSA-N propaneperoxoic acid Chemical compound CCC(=O)OO CZPZWMPYEINMCF-UHFFFAOYSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 125000004076 pyridyl group Chemical group 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Description
本発明は、担子菌由来ペルオキシダーゼを含有する化学発光測定試薬に関する。さらに詳しくは、ペルオキシダーゼを利用した臨床検査に使用できるペルオキシダーゼ化学発光測定試薬に関する。 The present invention relates to a chemiluminescence measuring reagent containing a basidiomycete-derived peroxidase. More specifically, the present invention relates to a reagent for measuring peroxidase chemiluminescence that can be used for clinical tests using peroxidase.
西洋ワサビ由来のペルオキシダーゼ（以下、「ＰＯＤ」という。）は、固相酵素免疫測定法（ＥＬＩＳＡ）、例えば、非競合法あるいは競合法、およびウエスタンブロッティング法などの各種の酵素免疫測定方法に利用される酵素のひとつとして、広く使用されている（例えば、特許文献１参照）。しかし、西洋ワサビ由来のＰＯＤは、原料としての西洋ワサビの栽培に長時間を要する上に、植物体を破壊し、多種多様な夾雑成分の中からＰＯＤを精製するという方法で製造されるため、その製造効率は高いとは言いがたく、大量生産が困難であった。その上、近年では、栽培効率の悪さや、需要の大きいバイオエタノール用穀物への転作などを理由に、ＰＯＤの原料としての西洋ワサビの供給不安が生じつつある状況が懸念されており、これに代わり得る酵素に対する潜在的なニーズは大きい。 Horseradish-derived peroxidase (hereinafter referred to as “POD”) is used in various enzyme immunoassay methods such as solid phase enzyme immunoassay (ELISA), for example, non-competitive or competitive methods, and Western blotting methods. It is widely used as one of the enzymes (see, for example, Patent Document 1). However, since POD derived from horseradish takes a long time to cultivate horseradish as a raw material, it is produced by a method of destroying the plant body and purifying POD from a wide variety of contaminated components. It was difficult to say that its production efficiency was high, and mass production was difficult. In addition, in recent years, there is concern about the situation where there is concern about the supply of horseradish as a raw material for POD, due to poor cultivation efficiency and conversion to high-demand bioethanol grains. The potential needs for alternative enzymes are great.
さらに、西洋ワサビ由来のＰＯＤには、多くのアイソザイムが存在するという問題も存在する。現在広く用いられている西洋ワサビ由来のＰＯＤの多くは、上述の通り低い生産効率の中で一定の価格で流通させるという制約があるため、多くのアイソザイムの混合物である場合がほとんどである。しかし、このようなＰＯＤを用いて各種の測定、例えば、酵素免疫測定を行った場合、異なる反応特性を有する各種アイソザイムの含有量が、ＰＯＤの製造ロットごとにばらつき、このことに起因して、安定した測定結果を得ることが困難になるという重大な問題を生じている。 Further, POD derived from horseradish has a problem that many isozymes exist. Most of the PODs derived from horseradish that are currently widely used have a restriction that they are distributed at a constant price within a low production efficiency as described above, and thus are mostly a mixture of many isozymes. However, when performing various measurements using such POD, for example, enzyme immunoassay, the content of various isozymes having different reaction characteristics varies from production lot to production lot of POD. There is a serious problem that it is difficult to obtain a stable measurement result.
前記のような西洋ワサビ由来のＰＯＤが有する問題を克服し得ることが期待されるＰＯＤとして、微生物由来のＰＯＤがある。微生物は短時間で大量に培養可能であり、微生物由来のＰＯＤは、植物体から精製を行う場合よりも格段に少ない手間で精製を行うことができる。また、遺伝子組換え技術を用いることにより、微生物宿主内での発現量を人為的に高めることも容易である。遺伝子組換え技術を利用すれば、目的とするＰＯＤのみを多量に発現させることができるため、アイソザイムの夾雑という問題も回避が容易であると同時に、そのＰＯＤを改変し、改良することも比較的容易である。このようなことから、微生物由来のＰＯＤは、西洋ワサビ由来のＰＯＤに代わり得る有望な酵素といえる。 POD derived from microorganisms is a POD expected to be able to overcome the problems of POD derived from horseradish as described above. Microorganisms can be cultivated in a large amount in a short time, and POD derived from microorganisms can be purified with much less labor than when purifying from a plant body. In addition, it is easy to artificially increase the expression level in a microbial host by using a gene recombination technique. If gene recombination technology is used, only the target POD can be expressed in a large amount. Therefore, it is easy to avoid the problem of isozyme contamination, and at the same time, it is relatively easy to modify and improve the POD. Easy. Therefore, it can be said that POD derived from microorganisms is a promising enzyme that can replace POD derived from horseradish.
しかし、微生物由来のＰＯＤを用いて、酵素免疫測定における性能を検討した知見は乏しい。公知の微生物由来ＰＯＤとしては、Arthromyces属由来のＰＯＤが知られ、遊離状態で、あるいは、抗体と結合させて標識抗体を調製し、その標識抗体を、測定物を介して固層に固定した状態で用いる測定系において、西洋ワサビ由来のＰＯＤよりも反応性が優れていることが報告されている（例えば、特許文献２参照）。また、過ヨウ素酸法を用いてArthromyces ramosus由来のＰＯＤで標識した抗体を用いたＥＬＩＳＡ法の系において、西洋ワサビ由来のＰＯＤ標識抗体を用いた場合よりも反応性が高いことが報告されている（例えば、非特許文献１参照）。しかし、Arthromyces属由来のＰＯＤの反応性の高さは、西洋ワサビ由来のＰＯＤを標識した抗体を用いた場合の４倍程度にとどまり、さらに、耐熱性にも問題があることが報告されている。 However, the knowledge which examined the performance in enzyme immunoassay using POD derived from microorganisms is scarce. Known POD derived from the genus Arthromyces is known as a microorganism-derived POD, and is prepared in a free state or by binding to an antibody to prepare a labeled antibody, and the labeled antibody is fixed to a solid layer via a measurement object It has been reported that the measurement system used in the present invention is superior in reactivity to POD derived from horseradish (see, for example, Patent Document 2). In addition, it has been reported that in an ELISA method using an antibody labeled with POD derived from Arthromyces ramosus using the periodic acid method, the reactivity is higher than when using a POD-labeled antibody derived from horseradish. (For example, refer nonpatent literature 1). However, the reactivity of POD derived from Arthromyces genus is only about 4 times higher than that obtained by using an antibody labeled with horseradish-derived POD, and it is reported that there is a problem in heat resistance. .
上記欠点を解決するために、本発明者らは、すでに担子菌Coprinus属由来のＰＯＤ（例えば、特許文献３、非特許文献２参照）を調製し、これを遊離状態で用いて、発色反応系または発光反応系における性能に関して、西洋ワサビ由来のＰＯＤの反応性との比較を試みている。その結果、２，２’−アジノビス（３−エチルベンゾチアゾリン−６−スルホン酸）（ＡＢＴＳ）を基質とした発色反応において、Coprinus属由来のＰＯＤは、西洋ワサビＰＯＤよりも９〜１０倍程度高活性であることを明らかにしている。また、ルミノールを基質とした発光反応系においては、化学発光増強剤であるｐ−ヨードフェノール等を添加しない場合、Coprinus属由来のＰＯＤは、西洋ワサビＰＯＤと比較して１００倍以上の発光強度が見られることを確認している。また、Coprinus属由来のＰＯＤで抗体を標識し、当該ＰＯＤ標識抗体を、化学発光増強剤を添加しない遊離状態で、ルミノールを基質とした発光反応に用いた場合にも、化学発光増強剤を添加した西洋ワサビ由来のＰＯＤ標識抗体に対して、同等もしくは同等以上の反応性を有することを確認している。また、耐熱性に関しても、Coprinus属由来ＰＯＤは、西洋ワサビ由来と同等あるいは同等以上の耐熱性を有しており、実用上問題とならないことも明らかにしている。 In order to solve the above disadvantages, the present inventors have already prepared a POD derived from the basidiomycete Coprinus genus (see, for example, Patent Document 3 and Non-Patent Document 2), and used this in a free state to produce a color reaction system. Or, in terms of performance in a luminescent reaction system, we are trying to compare it with the reactivity of POD derived from horseradish. As a result, in the color reaction using 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate, the POD derived from the genus Coprinus is about 9 to 10 times higher than the horseradish POD. It is revealed that it is active. In addition, in a luminescence reaction system using luminol as a substrate, when no chemiluminescence enhancer such as p-iodophenol is added, POD derived from the genus Coprinus has a luminescence intensity 100 times or more that of horseradish POD. Confirm that it can be seen. In addition, when the antibody is labeled with POD derived from Coprinus genus and the POD-labeled antibody is used in a luminescence reaction using luminol as a substrate in a free state without adding a chemiluminescence enhancer, a chemiluminescence enhancer is also added. It has been confirmed that the POD-labeled antibody derived from horseradish has equivalent or equivalent reactivity. In addition, regarding heat resistance, Coprinus genus-derived POD has the same or equivalent heat resistance as horseradish, and it has also been clarified that there is no practical problem.
しかしながら、Coprinus属由来のＰＯＤを含む微生物由来のＰＯＤは、従来の化学発光反応の条件下では、化学発光反応開始直後に、高い発光強度を示すものの、持続性が悪いという欠点を有している。そのため、安定状態で発光量を測定することが困難で、微生物由来のＰＯＤを西洋ワサビ由来のＰＯＤに代替し、酵素免疫測定等に利用するには発光持続性向上のための解決すべき課題が残されている。 However, PODs derived from microorganisms including PODs derived from the genus Coprinus show a high emission intensity immediately after the start of the chemiluminescence reaction under the conditions of the conventional chemiluminescence reaction, but have a drawback of poor sustainability. . Therefore, it is difficult to measure the amount of luminescence in a stable state, and there is a problem to be solved for improving the luminescence persistence in order to replace POD derived from microorganisms with POD derived from horseradish and use it for enzyme immunoassay and the like. It is left.
すなわち、化学発光強度が高く反応性・感度の優れた酵素免疫測定方法の開発が望まれており、特に、化学発光反応開始から初発時発光強度を長時間持続することができる化学発光試薬組成の提供が望まれている。 That is, development of an enzyme immunoassay method with high chemiluminescence intensity and excellent reactivity and sensitivity is desired, and in particular, a chemiluminescence reagent composition capable of maintaining the initial emission intensity for a long time from the start of the chemiluminescence reaction. Offer is desired.
本発明の課題は、化学発光反応開始から初発時発光強度を長時間持続することができる化学発光試薬組成を提供することにある。 An object of the present invention is to provide a chemiluminescent reagent composition capable of maintaining the initial emission intensity for a long time from the start of the chemiluminescent reaction.
本発明者らは、前記課題を解決するため鋭意検討を重ねた結果、特定の化学発光物質の含有量および酸化剤の含有量、並びに緩衝液の特定のｐＨを含む試薬を用いることにより、化学発光反応における担子菌由来ＰＯＤの初発時の発光強度を有効的に持続できることを知り、本発明を完成した。すなわち本発明は、以下に関する。
１）
（ａ）担子菌由来ペルオキシダーゼ
（ｂ）酸化剤
（ｃ）化学発光物質
（ｄ）緩衝液
を含んでなるペルオキシダーゼ化学発光測定試薬であって、化学発光反応開始時において、（ｂ）濃度０．０５〜１．０ｍＭ、（ｃ）濃度０．５〜５．０ｍＭで、かつ（ｄ）ｐＨが７．５〜９．５であり、
（ａ）が遊離状態のペルオキシダーゼである場合、（ｂ）および（ｃ）の濃度比が、（ｂ）濃度１に対して、（ｃ）濃度が１．０〜１０．０の範囲で、かつ、（ｄ）のｐＨが７．５〜９．５の範囲であり、
（ａ）が抗体に標識したペルオキシダーゼである場合、（ｂ）および（ｃ）の濃度比が、（ｂ）濃度１に対して、（ｃ）濃度が４．０〜２０．０の範囲で、かつ、（ｄ）ｐＨが８．０〜９．５の範囲であることを特徴とするペルオキシダーゼ化学発光測定試薬。
２）前記１）に記載のペルオキシダーゼ化学発光測定試薬において、（ａ）がCoprinus属由来ペルオキシダーゼで、（ｂ）が過酸化水素で、かつ（ｃ）がルミノールまたはその塩であることを特徴とするペルオキシダーゼ化学発光測定試薬。
３）前記１又は２）に記載のペルオキシダーゼ化学発光測定試薬において、ａ）が遊離状態のペルオキシダーゼであり、（ｄ）のｐＨが８．０の場合、（ｂ）濃度０．０５〜１．０ｍＭ、（ｃ）濃度０．５〜５．０ｍＭであり、（ｂ）および（ｃ）の濃度比が、（ｂ）濃度１に対して、（ｃ）濃度が１．０〜５．０の範囲であり、（ｄ）ｐＨが９．０〜９．５の範囲の場合、（ｂ）および（ｃ）の濃度比が、（ｂ）濃度１に対して、（ｃ）濃度が５．０〜１０．０の範囲であることを特徴とするペルオキシダーゼ化学発光測定試薬。
４）前記１又は２）に記載のペルオキシダーゼ化学発光測定試薬において、（ａ）が抗体に標識したペルオキシダーゼであって、（ｂ）濃度０．１１〜０．２２ｍＭ、（ｃ）濃度１．０〜３．０ｍＭであり、（ｂ）および（ｃ）の濃度比が、（ｂ）濃度１に対して、（ｃ）濃度が４．０〜２０．０の範囲であることを特徴とするペルオキシダーゼ化学発光測定試薬。
As a result of intensive studies in order to solve the above problems, the present inventors have found that chemicals can be obtained by using a reagent containing a specific chemiluminescent substance content and an oxidizing agent content, and a specific pH of a buffer solution. Knowing that the initial emission intensity of basidiomycete-derived POD in the luminescence reaction can be effectively sustained, the present invention has been completed. That is, the present invention relates to the following.
1)
(A) Basidiomycete-derived peroxidase (b) Oxidizing agent (c) Chemiluminescent substance (d) A peroxidase chemiluminescence measuring reagent comprising a buffer solution. -1.0 mM, (c) concentration 0.5-5.0 mM, and (d) pH 7.5-9.5,
When (a) is a free peroxidase, the concentration ratio of (b) and (c) is (c) in the range of 1.0 to 10.0 with respect to (b) concentration 1, and , (D) has a pH in the range of 7.5 to 9.5,
When (a) is a peroxidase labeled with an antibody, the concentration ratio of (b) and (c) is in the range of (c) concentration of 4.0 to 20.0 with respect to (b) concentration 1, (D) A reagent for measuring peroxidase chemiluminescence, wherein the pH is in the range of 8.0 to 9.5.
2) The peroxidase chemiluminescence measuring reagent according to 1) above, wherein (a) is a Coprinus genus peroxidase, (b) is hydrogen peroxide, and (c) is luminol or a salt thereof. Peroxidase chemiluminescence reagent.
3) In the reagent for measuring peroxidase chemiluminescence as described in 1 or 2) above, when a) is a free peroxidase and the pH of (d) is 8.0, (b) a concentration of 0.05 to 1.0 mM (C) concentration is 0.5 to 5.0 mM, and the concentration ratio of (b) and (c) is in the range of (c) concentration of 1.0 to 5.0 with respect to (b) concentration 1 (D) When the pH is in the range of 9.0 to 9.5, the concentration ratio of (b) and (c) is (b) concentration 1 with respect to (c) concentration 5.0 to A reagent for measuring peroxidase chemiluminescence, which is in the range of 10.0.
4) The peroxidase chemiluminescence measuring reagent according to 1 or 2) above, wherein (a) is a peroxidase labeled on an antibody, and (b) a concentration of 0.11 to 0.22 mM, (c) a concentration of 1.0 to Peroxidase chemistry, wherein the concentration ratio of (b) and (c) is in the range of 4.0 to 20.0 with respect to (b) concentration 1. Luminescence measuring reagent.
本発明により、担子菌由来ＰＯＤを用いて化学発光測定をする際に、初発時の発光強度を有効的に持続できる試薬を提供することができる。 According to the present invention, when chemiluminescence measurement is performed using a basidiomycete-derived POD, a reagent capable of effectively maintaining the luminescence intensity at the first occurrence can be provided.
（担子菌由来ＰＯＤ）
本発明に使用するＰＯＤとしては、担子菌由来のＰＯＤが挙げられる。担子菌に属する微生物としては、例えば、Coprinus属、Uredinales属、Auriculariales属、Agaricales属等が挙げられる。中でも、Coprinus属由来のＰＯＤは、西洋ワサビ由来のＰＯＤと比較して、同等もしくは同等以上の優れた酵素化学的性質（至適ｐＨ、ｐＨ安定性、至適温度、温度安定性）を有しており、しかも、２，２’−アジノビス（３−エチルベンゾチアゾリン−６−スルホン酸）（ＡＢＴＳ）を基質とした発色反応において、西洋ワサビ由来のＰＯＤよりも比活性（Ｕ／ｍｇ）が９〜１０倍程度高いため好ましい。Coprinus属に属する微生物の例としては、Coprinus cinereus（NBRC30114）、Coprinus macrorhizus（ATCC20120）、Coprinellus disseminatus、Coprinus comatus（ATCC12640）、Coprinus clastophyllus、Coprinus alkalinus、Coprinus amphibius、Coprinus micaceus、Coprinus atramentarius、Coprinus luteocephalus、Coprinus trisporus、Coprinus sclerotiger、Coprinus domesticus、Coprinus stercorarius、Coprinus radiatus等が挙げられる。なお、NBRCは、独立行政法人製品評価技術基盤機構生物遺伝資源部門、ATCCは、Ａｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎを示す。
(Basidiomycete-derived POD)
Examples of the POD used in the present invention include basidiomycete-derived POD. Examples of the microorganism belonging to the basidiomycete include the genus Coprinus, the genus Uredinales, the genus Auriculariales, the genus Agaricales, and the like. Among them, POD derived from the genus Coprinus has excellent enzyme chemical properties (optimal pH, pH stability, optimal temperature, temperature stability) that are equivalent to or higher than those derived from horseradish. Moreover, in the color reaction using 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a substrate, the specific activity (U / mg) is 9 more than POD derived from horseradish. It is preferable because it is about 10 times higher. Examples of microorganisms belonging to the genus Coprinus include Coprinus cinereus (NBRC30114), Coprinus macrorhizus (ATCC20120), Coprinellus disseminatus, Coprinus comatus (ATCC12640), Coprinus clastophyllus, Coprinus alkalinus, Coprinus amphibius, Coprinus micaceus, Coprinus micaceus, Coprius micaceus, Colut Examples include trisporus, Coprinus sclerotiger, Coprinus domesticus, Coprinus stercorarius, Coprinus radiatus. In addition, NBRC indicates the National Institute for Product Evaluation and Technology Biological Resource Department, and ATCC indicates the American Type Culture Collection.
Coprinus属由来ＰＯＤは、天然のものであってもよく、耐熱性向上や基質特異性向上、その他の何らかの１以上の変異を人為的に導入したものであってもよく、キメラタンパク質等であってもよい。また、市販のCoprinus属由来ＰＯＤを用いてもよい。測定上支障がない範囲において、アイソザイムを含む複数のＰＯＤを共に使用することもできるが、測定の安定性のためには、単一精製したものを用いることが好ましい。 Coprinus genus-derived POD may be naturally occurring, may have improved heat resistance, improved substrate specificity, or may be artificially introduced with one or more other mutations, such as a chimeric protein. Also good. Moreover, you may use commercially available Coprinus genus POD. A plurality of PODs containing isozymes can be used together as long as there is no problem in measurement, but it is preferable to use a single purified product for the stability of measurement.
また、分類学上Coprinus属に属さない微生物であっても、例えば、Coprinus属に近縁の微生物由来のＰＯＤや、Coprinus属由来のＰＯＤとアミノ酸配列が近似するもので、本発明の酵素免疫測定において同様の反応性を示すＰＯＤ等も用いることができる。 In addition, even in the case of microorganisms that do not belong to the genus Coprinus, for example, PODs derived from microorganisms closely related to the genus Coprinus or PODs derived from the genus Coprinus are similar in amino acid sequence, and the enzyme immunoassay of the present invention POD or the like having the same reactivity can be used.
本発明に使用するＰＯＤは、後述する最終反応溶液中において、遊離状態または各種抗体を標識した状態で用いることができる。ＰＯＤへ抗体を標識（架橋）する方法としては、公知の各種の標識方法を用いることができ、例えば、一般的に知られている方法として、グルタルアルデヒド法、過ヨウ素酸法、マレイミド法、ピリジルジスルフィド法、イソシアネート架橋法、ベンゾキノン架橋法等が挙げられる。特に、マレイミド法は、重合体形成の有無、抗原、抗体、酵素の活性維持、さらには標識効率の点で好適である。 The POD used in the present invention can be used in a free state or a state in which various antibodies are labeled in the final reaction solution described later. As a method for labeling (crosslinking) an antibody to POD, various known labeling methods can be used. For example, generally known methods include glutaraldehyde method, periodic acid method, maleimide method, pyridyl Examples thereof include a disulfide method, an isocyanate crosslinking method, and a benzoquinone crosslinking method. In particular, the maleimide method is suitable in terms of the presence or absence of polymer formation, the maintenance of the activity of antigens, antibodies and enzymes, and the efficiency of labeling.
（酸化剤）
本発明に使用する酸化剤は、無機過酸化物として、例えば、過酸化水素、過ホウ酸塩、次亜塩素酸塩等が挙げられ、有機過酸化物として、例えば、過酢酸、過プロピオン酸等が挙げられる。取扱い易さから、過酸化水素を用いるのが好ましい。
(Oxidant)
Examples of the oxidizing agent used in the present invention include inorganic peroxides such as hydrogen peroxide, perborate and hypochlorite, and organic peroxides such as peracetic acid and perpropionic acid. Etc. From the viewpoint of ease of handling, it is preferable to use hydrogen peroxide.
酸化剤の含有量は、用いる化学発光物質の種類、濃度または適用する測定方法等によって適宜設定すればよいが、初発時発光強度を高い値で維持するために、最終反応溶液中の濃度を０．０５〜１．０ｍＭの濃度の範囲に保つ必要がある。前記濃度の範囲を下回る場合、初発時発光強度が減少し、実用的な測定感度を得ることができなくなる。一方、前記濃度の範囲を上回る場合、化学発光持続性が悪くなってしまう。 The content of the oxidizing agent may be appropriately set depending on the type and concentration of the chemiluminescent substance to be used or the measurement method to be applied. However, in order to maintain the initial emission intensity at a high value, the concentration in the final reaction solution is 0. It is necessary to keep the concentration in the range of 0.05 to 1.0 mM. When the concentration is below the range, the initial emission intensity decreases and practical measurement sensitivity cannot be obtained. On the other hand, when the concentration exceeds the range, the chemiluminescence persistence deteriorates.
（化学発光物質）
本発明に使用する化学発光物質としては、公知の各種化学発光物質を用いることができ、例えば、２，３−ジヒドロ−１，４−フタラジンジオン化合物を用いることができる。前記化学発光物質の具体的な例としては、例えば、ルミノール、イソルミノール、Ｎ−エチルイソルミノール、Ｎ−（４−アミノブチル）−Ｎ−エチルイソルミノールヘミサクシミド、Ｎ−（６−アミノヘキシル）−Ｎ−エチルイソルミノール等、あるいは、これらの金属塩が挙げられる。金属塩としては、アルカリ金属塩、アルカリ土類金属塩等を用いることができる。アルカリ金属塩としては、例えば、ナトリウム塩、カリウム塩等が挙げられ、アルカリ土類金属塩としては、カルシウム塩、マグネシウム塩等が挙げられる。例えば、ルミノールまたはルミノール金属塩が安定性や発光量子収率の点で好ましい。ルミノール金属塩としては、ナトリウム塩を用いることができる。
(Chemiluminescent substance)
As the chemiluminescent substance used in the present invention, various known chemiluminescent substances can be used. For example, 2,3-dihydro-1,4-phthalazinedione compounds can be used. Specific examples of the chemiluminescent substance include, for example, luminol, isoluminol, N-ethylisoluminol, N- (4-aminobutyl) -N-ethylisoluminol hemisuccimide, N- (6-aminohexyl). ) -N-ethylisoluminol and the like, or metal salts thereof. As the metal salt, an alkali metal salt, an alkaline earth metal salt, or the like can be used. Examples of the alkali metal salt include sodium salt and potassium salt, and examples of the alkaline earth metal salt include calcium salt and magnesium salt. For example, luminol or a luminol metal salt is preferable in terms of stability and emission quantum yield. As the luminol metal salt, a sodium salt can be used.
化学発光物質の含有量は、用いる酸化剤の種類、濃度または適用する測定方法等によって適宜設定すればよいが、初発時発光強度を高い値で維持するために、最終反応溶液中の濃度を０．５〜５．０ｍＭの濃度の範囲に保つ必要がある。前記濃度の範囲を下回る場合、初発時発光強度が減少し、実用的な測定感度を得ることができなくなる。一方、前記濃度の範囲を上回る場合、高濃度の化学発光物質により、かえって化学発光反応が阻害され、実用的な測定感度を得ることができなくなる。 The content of the chemiluminescent substance may be appropriately set depending on the kind and concentration of the oxidizing agent to be used or the measurement method to be applied. However, in order to maintain the initial emission intensity at a high value, the concentration in the final reaction solution is 0. It is necessary to keep the concentration within the range of 5 to 5.0 mM. When the concentration is below the range, the initial emission intensity decreases and practical measurement sensitivity cannot be obtained. On the other hand, when the concentration exceeds the range, the chemiluminescent reaction is inhibited by the high concentration of the chemiluminescent substance, so that practical measurement sensitivity cannot be obtained.
（緩衝液）
本発明に使用する緩衝液は、特に限定されることはなく、例えば、酢酸緩衝液、クエン酸緩衝液、リン酸緩衝液、トリス緩衝液、グッド緩衝液、ホウ酸緩衝液、グリシン緩衝液、ピロリン酸緩衝液、アンモニウム緩衝液、炭酸緩衝液等が挙げられる。緩衝作用が優れた緩衝液であればよい。
(Buffer solution)
The buffer used in the present invention is not particularly limited. For example, acetate buffer, citrate buffer, phosphate buffer, Tris buffer, Good buffer, borate buffer, glycine buffer, Examples include pyrophosphate buffer, ammonium buffer, and carbonate buffer. Any buffer solution with excellent buffering action may be used.
緩衝液のｐＨは、用いる化学発光物質もしくは酸化剤の種類、濃度または適用する測定方法等によって適宜設定すればよいが、初発時発光強度を高い値で維持するために、最終反応溶液中のｐＨを７．５〜１０．０の範囲に保つことが好ましい。前記ｐＨの範囲を下回る場合、初発時発光強度が減少し、実用的な測定感度を得ることができなくなる。一方、前記ｐＨの範囲を上回る場合、化学発光持続性が悪くなってしまう。なお、緩衝液のｐＨは、緩衝剤の水溶液に無機アルカリあるいは無機酸の水溶液を加えることで調整することができる。 The pH of the buffer solution may be set as appropriate depending on the type and concentration of the chemiluminescent substance or oxidant to be used, or the measurement method to be applied, but in order to maintain the initial emission intensity at a high value, the pH in the final reaction solution Is preferably kept in the range of 7.5 to 10.0. When the pH is below the range, the initial emission intensity decreases and practical measurement sensitivity cannot be obtained. On the other hand, when it exceeds the pH range, the chemiluminescence persistence deteriorates. The pH of the buffer solution can be adjusted by adding an aqueous solution of an inorganic alkali or an inorganic acid to the aqueous solution of the buffer.
（最終反応溶液）
前記のＰＯＤ、酸化剤、化学発光物質および緩衝液を含んでなる最終反応溶液中で、化学発光反応が起こる。初発時発光強度を高い値で維持するために、特に重要な因子として、最終反応溶液における酸化剤および化学発光物質の濃度、酸化剤および化学発光物質の濃度比、ｐＨが挙げられる。これらを適正に選択して組み合わせることにより、高い発光持続性を実現することができる。
(Final reaction solution)
A chemiluminescent reaction takes place in the final reaction solution comprising the POD, oxidant, chemiluminescent material and buffer. In order to maintain the initial emission intensity at a high value, particularly important factors include the concentration of the oxidizing agent and the chemiluminescent substance in the final reaction solution, the concentration ratio of the oxidizing agent and the chemiluminescent substance, and the pH. By appropriately selecting and combining these, high light emission sustainability can be realized.
最終反応溶液における化学発光物質ならびに酸化剤の濃度および緩衝液のｐＨは、上述したとおり、酸化剤濃度０．０５〜１．０ｍＭ、化学発光物質濃度０．５〜５．０ｍＭで、かつｐＨ７．５〜１０．０の範囲である。さらに、酸化剤および化学発光物質の濃度比を、酸化剤１に対して、化学発光物質を１．０〜２０．０に保つ必要がある。最終反応溶液における各因子が前記範囲内であれば、化学発光反応開始１０分後において７０％以上の初発時発光強度を持続し、安定性、反応性および感度の優れた酵素免疫測定に供することができる。特に、酸化剤および化学発光物質の濃度比を上記の範囲に保つことが重要である。該濃度比が１．０を下回る場合は、化学発光反応開始１０分後における発光強度は、初発時発光強度の７０％を下回り、測定が極めて困難となる。一方、濃度比が２０．０を上回る場合は、化学発光反応を開始後しばらくしてから急激に発光反応が進行してしまい、安定性を欠くため、測定が極めて困難となる。化学発光反応開始５分後における発光強度が、初発時発光強度の２５０％を上回る場合は、臨床検査等への応用は不向きとなる。 As described above, the chemiluminescent substance and the oxidizing agent in the final reaction solution and the buffer solution have an oxidizing agent concentration of 0.05 to 1.0 mM, a chemiluminescent substance concentration of 0.5 to 5.0 mM, and a pH of 7. It is in the range of 5 to 10.0. Furthermore, the concentration ratio of the oxidant and the chemiluminescent substance needs to be kept at 1.0 to 20.0 with respect to the oxidant 1. If each factor in the final reaction solution is within the above range, the initial luminescence intensity of 70% or more is maintained 10 minutes after the start of the chemiluminescence reaction, and the enzyme immunoassay is excellent in stability, reactivity and sensitivity. Can do. In particular, it is important to maintain the concentration ratio of the oxidizing agent and the chemiluminescent substance within the above range. When the concentration ratio is less than 1.0, the luminescence intensity 10 minutes after the start of the chemiluminescence reaction is less than 70% of the initial luminescence intensity, which makes measurement extremely difficult. On the other hand, when the concentration ratio exceeds 20.0, the luminescence reaction proceeds abruptly after a while after the start of the chemiluminescence reaction and lacks stability, making measurement extremely difficult. When the luminescence intensity at 5 minutes after the start of the chemiluminescence reaction exceeds 250% of the initial luminescence intensity, application to a clinical test or the like is not suitable.
なお、用いるＰＯＤが遊離状態であるか、抗体に標識した状態であるかの違いにより反応性が異なるため、前記の範囲で適宜選択するのが好ましく、例えば、遊離状態のＰＯＤを用いる場合、ｐＨが７．５〜９．５で、かつ、酸化剤および化学発光物質の濃度比を、酸化剤１に対して、化学発光物質を１．０〜１０．０とするのが好ましく、抗体に標識したＰＯＤを用いる場合、ｐＨが８．０〜９．５で、かつ酸化剤および化学発光物質の濃度比を、酸化剤１に対して、化学発光物質を４．０〜２０．０とするのが好ましい。 The reactivity varies depending on whether the POD to be used is in a free state or in a state labeled with an antibody. Therefore, it is preferable to select appropriately within the above range. For example, when using a free state POD, Is preferably 7.5 to 9.5, and the concentration ratio of the oxidizing agent and the chemiluminescent substance is preferably 1.0 to 10.0 with respect to the oxidizing agent 1, and the antibody is labeled. In the case of using the POD, the pH is 8.0 to 9.5, and the concentration ratio of the oxidant and the chemiluminescent substance is set to 4.0 to 20.0 with respect to the oxidant 1 with the chemiluminescent substance being 4.0 to 20.0. Is preferred.
以下、実施例により、本発明をさらに具体的に説明する。ただし、本発明の技術的範囲は、それらの例により何ら限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by these examples.
［遊離状態のＰＯＤを用いた緩衝液の検討］
緩衝液の化学発光反応の発光持続性に対する影響を検討するため、各種緩衝液を調製し、実験に供することで比較検討を行った。１００、２００、３００ｍＭトリス緩衝液、１００、１５０ｍＭリン酸緩衝液および１００、１５０ｍＭクエン酸リン酸緩衝液を常法に従い調製した。ＰＯＤとして、市販のＰＯＤ（Ｒｏｃｈｅ社製）を用い、約２０ｍｇ／ｍｌとなるように、酵素希釈用緩衝液（０．１５％ Ｔｒｉｔｏｎ Ｘ−４０５を含む０．０５Ｍ リン酸緩衝液（ｐＨ６．４））に溶解し、さらに同希釈液で１００万〜５，０００万倍にＰＯＤを希釈して試験に供した。
[Examination of buffer solution using POD in free state]
In order to examine the influence of the buffer solution on the luminescence persistence of the chemiluminescence reaction, various buffer solutions were prepared and subjected to a comparative study. 100, 200, 300 mM Tris buffer, 100, 150 mM phosphate buffer and 100, 150 mM citrate phosphate buffer were prepared according to a conventional method. As a POD, a commercially available POD (manufactured by Roche) was used, and a buffer solution for enzyme dilution (0.05 M phosphate buffer (pH 6.4 containing 0.15% Triton X-405) was adjusted to about 20 mg / ml. )), POD was diluted 1 million to 50 million times with the same diluent, and used for the test.
化学発光反応は、最終反応溶液中のルミノール（Ｓｉｇｍａ社製）および過酸化水素（和光純薬社製）の濃度が、表１に示す濃度となるよう、あらかじめ各緩衝液にルミノールおよび過酸化水素を溶解し、希釈した前記ＰＯＤを添加して行った。初発時発光強度は、初発時（反応開始直後０分）、反応開始後５、１０、１５分における各発光量（１秒間の平均発光積算量）を、ＭｉｃｒｏＬｕｍａｔＰｌｕｓ ＬＢ ９６Ｖ（Ｂｅｒｔｈｏｌｄ社製）を用いて測定し、初発時の発光量を１００％とする相対値として算出した。この結果を表１に示す。 In the chemiluminescence reaction, luminol and hydrogen peroxide were previously added to each buffer solution so that the concentrations of luminol (manufactured by Sigma) and hydrogen peroxide (manufactured by Wako Pure Chemical Industries) in the final reaction solution were the concentrations shown in Table 1. And the diluted POD was added. As for the initial luminescence intensity, MicroLumatPlus LB 96V (manufactured by Berthold) was used for each luminescence amount (average luminescence integrated amount for 1 second) at the time of first lapse (0 minutes immediately after the start of reaction), 5, 10, 15 minutes after the start of reaction. And calculated as a relative value with the initial light emission amount being 100%. The results are shown in Table 1.
表１に示すとおり、用いる緩衝液の種類によらず、最終反応溶液のｐＨ、ルミノール濃度および過酸化水素濃度を適正に選択して組み合わせることにより、化学発光反応開始１０分後においても、相対発光量が７０％以上に維持されることが確認された。 As shown in Table 1, regardless of the type of buffer used, relative light emission can be achieved even after 10 minutes from the start of the chemiluminescence reaction by appropriately selecting and combining the pH, luminol concentration and hydrogen peroxide concentration of the final reaction solution. It was confirmed that the amount was maintained at 70% or more.
［遊離状態のＰＯＤを用いた最終反応溶液中の各濃度の検討］
ＰＯＤの発光持続性に、ルミノールおよび過酸化水素の濃度ならびに両者の濃度比が与える影響を検討した。化学発光反応は、２００ｍＭ、ｐＨ８．５〜９．５のトリス緩衝液にルミノールおよび過酸化水素の終濃度が表２、３に示す濃度となるよう、あらかじめ各緩衝液に溶解し、同じく表２、３に示すような最終反応溶液濃度になるように調製した試薬に、実施例１と同様に希釈した市販のＰＯＤを添加することで行った。初発時発光強度は、実施例１の方法と同様に測定した。結果を表２、３に示す。
[Examination of each concentration in the final reaction solution using POD in free state]
The effect of the concentration of luminol and hydrogen peroxide and the concentration ratio of both on the luminescence persistence of POD was examined. The chemiluminescence reaction was previously dissolved in each buffer solution so that the final concentrations of luminol and hydrogen peroxide were as shown in Tables 2 and 3 in a Tris buffer solution of 200 mM, pH 8.5 to 9.5. 3 was performed by adding a commercially available POD diluted to the reagent prepared so as to have a final reaction solution concentration as shown in 3, in the same manner as in Example 1. The initial emission intensity was measured in the same manner as in the method of Example 1. The results are shown in Tables 2 and 3.
表２、３に示すとおり、最終反応溶液のｐＨ、ルミノール濃度および過酸化水素濃度を適正に選択して組み合わせることにより、発光反応開始後も高い発光持続性を保つことが確認された。 As shown in Tables 2 and 3, it was confirmed that high luminescence persistence was maintained even after the start of the luminescence reaction by appropriately selecting and combining the pH, luminol concentration and hydrogen peroxide concentration of the final reaction solution.
遊離状態のＰＯＤを用いて化学発光測定に供した場合、緩衝液のｐＨにもよるが、最終反応溶液中に含まれるルミノールおよび過酸化水素の濃度範囲が、それぞれ０．５〜５．０ｍＭおよび０．０５〜１．０ｍＭの範囲内で、かつ、ルミノール／過酸化水素の濃度比が、１．０〜１０．０において、高い発光安定性および発光持続性が保たれることがわかる。また、緩衝液のｐＨが８．５の場合、ルミノール／過酸化水素の濃度比は、１．０〜５．０が好ましく、緩衝液のｐＨが９．０〜９．５の場合、濃度比は５．０〜１０．０が好ましい。濃度比が１０を越える場合、反応直後に急激に発光量が増加し、安定性が保たれなくなってしまうため、正確な値を測定できなくなってしまう。一方、濃度比が低くなるにつれ、化学発光持続性が悪くなってしまい、濃度比が１を下回る場合は、実測に耐えられなくなってしまう。 When subjected to chemiluminescence measurement using a free POD, the concentration range of luminol and hydrogen peroxide contained in the final reaction solution is 0.5 to 5.0 mM and 0.5 mM, respectively, depending on the pH of the buffer solution. It can be seen that high luminescence stability and luminescence persistence are maintained when the concentration ratio of luminol / hydrogen peroxide is within the range of 0.05 to 1.0 mM and the concentration ratio of luminol / hydrogen peroxide is 1.0 to 10.0. When the pH of the buffer is 8.5, the concentration ratio of luminol / hydrogen peroxide is preferably 1.0 to 5.0, and when the pH of the buffer is 9.0 to 9.5, the concentration ratio Is preferably 5.0 to 10.0. When the concentration ratio exceeds 10, the amount of luminescence increases rapidly immediately after the reaction and the stability cannot be maintained, so that an accurate value cannot be measured. On the other hand, as the concentration ratio decreases, the chemiluminescence persistence deteriorates, and when the concentration ratio is less than 1, the actual measurement cannot be endured.
［ＰＯＤ標識マウスＩｇＧ抗体を用いた最終反応溶液中の各濃度の検討］
次に、ＰＯＤ標識抗体を用いた際の、最終反応溶液の成分条件を検討した。
１．抗マウスＩｇＧ抗体へのＰＯＤ標識
抗マウスＩｇＧ抗体へのＰＯＤ標識を、以下のように行った。
１）ＩｇＧ−ＳＨの調製
ヤギ抗マウス抗体（ＩｇＧ）を、０．１５Ｍ ＮａＣｌおよび１０ｍＭ ＥＤＴＡを含む０．１Ｍ リン酸ナトリウム緩衝液（ｐＨ６．４）に溶液置換し、１〜１０ｍｇ／ｍｌとなるように調製した。５００ｍＭ システアミン（Ｓｉｇｍａ社製；製品コード：Ｍ９７６８−２５Ｇ）（システアミン６ｍｇを０．１５Ｍ ＮａＣｌおよび１０ｍＭ ＥＤＴＡを含む０．１Ｍ リン酸ナトリウム緩衝液（ｐＨ６．４）０．１ ｍｌに溶解）を調製し、前記の抗体溶液に１／１０（ｖ／ｖ）量加え、３７℃にて１．５時間インキュベーションした。過剰なシステアミンを透析あるいはゲルろ過カラムＳｅｐｈａｄｅｘ Ｇ−２５（ＧＥ社製；製品コード：１７−００３３−０１、平衡化液：０．１５Ｍ ＮａＣｌおよび１０ｍＭ ＥＤＴＡを含む０．１Ｍ リン酸ナトリウム緩衝液（ｐＨ６．８）、溶出液：平衡化液と同様、カラムサイズ：１０ｍｌ、重力による溶出）により除去した。
２）マレイミド化ＰＯＤの調製
市販のＰＯＤ（Ｒｏｃｈｅ社製）を１０〜３０ｍｇ／ｍｌとなるように、０．１５Ｍ ＮａＣｌおよび１０ｍＭ ＥＤＴＡを含む０．１Ｍ リン酸ナトリウム緩衝液（ｐＨ７．４）に溶解した。１００ｍＭ ＳＭＣＣ（Ｐｉｅｒｃｅ社製；製品コード、２２３６０）（３．３４ｍｇを０．１ｍｌのＤＭＳＯに溶解）を調製し、直ちにＰＯＤのモル濃度の５倍量を加えた。室温で３０分間インキュベーションし、前記の条件に従って、ゲルろ過カラムＳｅｐｈａｄｅｘ Ｇ−２５に供し、過剰なＳＭＣＣを除去した。酵素濃度を、４０３ｎｍにおける吸光度測定値から、ε４０３ｎｍ＝８．３３×１０４ｌ／（ｍｏｌ・ｃｍ）を用いて求めた。
３）マレイミド化ＰＯＤによる抗体の標識（架橋）および標識抗体の精製
前記のマレイミド化ＰＯＤおよびＳＨ基還元抗体（溶媒は０．１５Ｍ ＮａＣｌおよび１０ｍＭ ＥＤＴＡを含む０．１Ｍ リン酸ナトリウム緩衝液（ｐＨ６．８））を、酵素：抗体のモル比が１０：１となるように混合した。４℃で６時間以上（一晩）インキュベーションした後、標識（架橋）後の反応溶液を、０．５Ｍ ＮａＣｌを含む０．１Ｍ リン酸ナトリウム緩衝液（ｐＨ６．８）で平衡化したＳｕｐｅｒｄｅｘ ２００ １０／３００ＧＬ（ＧＥ社製；製品コード：１７−５１７５−０１、溶出：０．５Ｍ ＮａＣｌを含む０．１Ｍ リン酸ナトリウム緩衝液（ｐＨ６．８）、カラムサイズ：約２４ｍｌ、流速：０．５ｍｌ／ｍｉｎ）に供することにより、抗体と結合されなかった酵素を除去して、ＰＯＤ標識抗マウスＩｇＧ（ＩｇＧのＰＯＤ標識抗体、ＰＯＤ−ＩｇＧ）を得た。
[Examination of each concentration in the final reaction solution using POD-labeled mouse IgG antibody]
Next, the component conditions of the final reaction solution when using a POD-labeled antibody were examined.
1. POD labeling to anti-mouse IgG antibody POD labeling to anti-mouse IgG antibody was performed as follows.
1) Preparation of IgG-SH Solution replacement of goat anti-mouse antibody (IgG) with 0.1 M sodium phosphate buffer (pH 6.4) containing 0.15 M NaCl and 10 mM EDTA results in 1 to 10 mg / ml It was prepared as follows. 500 mM cysteamine (manufactured by Sigma; product code: M9768-25G) (6 mg of cysteamine dissolved in 0.1 ml of 0.1 M sodium phosphate buffer (pH 6.4) containing 0.15 M NaCl and 10 mM EDTA) was prepared. 1/10 (v / v) amount was added to the antibody solution and incubated at 37 ° C. for 1.5 hours. Excess cysteamine was dialyzed or gel filtration column Sephadex G-25 (manufactured by GE; product code: 17-0033-01, equilibration solution: 0.1 M sodium phosphate buffer (pH 6) containing 0.15 M NaCl and 10 mM EDTA. 8), eluent: same as the equilibration liquid, column size: 10 ml, elution by gravity).
2) Preparation of maleimidated POD Commercially available POD (Roche) was dissolved in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.15 M NaCl and 10 mM EDTA so as to be 10 to 30 mg / ml. did. 100 mM SMCC (Pierce; product code: 22360) (3.34 mg dissolved in 0.1 ml DMSO) was prepared and immediately added 5 times the molar concentration of POD. After incubating at room temperature for 30 minutes, according to the above conditions, the gel filtration column Sephadex G-25 was used to remove excess SMCC. The enzyme concentration was determined from the measured absorbance at 403 nm using ε403 nm = 8.33 × 10 4 l / (mol · cm).
3) Labeling (crosslinking) of antibody with maleimidated POD and purification of labeled antibody The maleimidated POD and SH group-reduced antibody (solvent is 0.1M sodium phosphate buffer (pH 6. 8)) was mixed so that the molar ratio of enzyme: antibody was 10: 1. After incubation at 4 ° C. for 6 hours or more (overnight), the reaction solution after labeling (crosslinking) was equilibrated with 0.1 M sodium phosphate buffer (pH 6.8) containing 0.5 M NaCl, Superdex 200 10 / 300GL (manufactured by GE; product code: 17-5175-01, elution: 0.1 M sodium phosphate buffer (pH 6.8) containing 0.5 M NaCl, column size: about 24 ml, flow rate: 0.5 ml / min), the enzyme that was not bound to the antibody was removed, and POD-labeled anti-mouse IgG (IgG POD-labeled antibody, POD-IgG) was obtained.
２．ＥＬＩＳＡ法におけるＰＯＤ−ＩｇＧを用いたマウスＩｇＧの検出
８％ ＮａＣｌおよび０．２％ ＫＣｌを含む０．０１Ｍ リン酸ナトリウム緩衝液（ｐＨ７．４）で、１μｇ／ｍｌの濃度に調製したマウスＩｇＧ溶液（抗ヒトトランスフェリンモノクローナル抗体（バイオマトリックス研究所社製））を用い、定法により、９６ウェルマイクロタイタープレート中にマウスＩｇＧを固相化した。その後、０．０５％ Ｔｗｅｅｎ ２０および１５０ｍＭ ＮａＣｌを含む５０ｍＭ トリス塩酸緩衝液（ｐＨ ７．５）で３回洗浄した。ここに、１％スキムミルク、０．０５％ Ｔｗｅｅｎ ２０および１５０ｍＭ ＮａＣｌを含む５０ｍＭ トリス塩酸緩衝液（ｐＨ ７．５）を用い、定法により、９６ウェルマイクロタイタープレートをブロッキングした。その後、０．０５％ Ｔｗｅｅｎ ２０および１５０ｍＭ ＮａＣｌを含む５０ｍＭ トリス塩酸緩衝液（ｐＨ ７．５）で３回洗浄した。次に、０．１％スキムミルク、０．０５％ Ｔｗｅｅｎ２０および１５０ｍＭ ＮａＣｌを含む５０ｍＭ トリス塩酸緩衝液（ｐＨ ７．５）で、１０〜５０ｎｇ／ｍｌの濃度に調製した前記ＰＯＤ標識抗体（ＰＯＤ−ＩｇＧ）を添加し、室温で１時間反応させた。その後、０．０５％ Ｔｗｅｅｎ ２０および１５０ｍＭ ＮａＣｌを含む５０ｍＭ トリス塩酸緩衝液（ｐＨ ７．５）で３回洗浄した。
2. Detection of mouse IgG using POD-IgG in ELISA method Mouse IgG solution prepared to a concentration of 1 μg / ml with 0.01 M sodium phosphate buffer (pH 7.4) containing 8% NaCl and 0.2% KCl Using a (anti-human transferrin monoclonal antibody (manufactured by Biomatrix Laboratories)), mouse IgG was immobilized on a 96-well microtiter plate by a conventional method. Then, it was washed 3 times with 50 mM Tris-HCl buffer (pH 7.5) containing 0.05% Tween 20 and 150 mM NaCl. A 96-well microtiter plate was blocked by a conventional method using 50 mM Tris-HCl buffer (pH 7.5) containing 1% skim milk, 0.05% Tween 20 and 150 mM NaCl. Then, it was washed 3 times with 50 mM Tris-HCl buffer (pH 7.5) containing 0.05% Tween 20 and 150 mM NaCl. Next, the POD-labeled antibody (POD-IgG) prepared at a concentration of 10 to 50 ng / ml with 50 mM Tris-HCl buffer (pH 7.5) containing 0.1% skim milk, 0.05% Tween 20 and 150 mM NaCl. ) Was added and allowed to react for 1 hour at room temperature. Then, it was washed 3 times with 50 mM Tris-HCl buffer (pH 7.5) containing 0.05% Tween 20 and 150 mM NaCl.
化学発光反応は、１００ｍＭ、ｐＨ８．０〜９．０のトリス緩衝液あるいは２００ｍＭ、ｐＨ９．０〜９．５のトリス緩衝液に、ルミノールおよび過酸化水素の終濃度が表４に示す濃度となるよう、あらかじめ各緩衝液に溶解し、同じく表４に示すような最終反応溶液濃度になるように調製した試薬を、上記のように調製した９６ウェルマイクロタイタープレートに添加し、一定時間反応させることで行った。初発時発光強度は、実施例１の方法と同様に測定した。結果を、表４に示す。 In the chemiluminescence reaction, the final concentrations of luminol and hydrogen peroxide are as shown in Table 4 in a Tris buffer solution of 100 mM, pH 8.0 to 9.0 or a Tris buffer solution of 200 mM, pH 9.0 to 9.5. As shown in Table 4, add the reagent prepared in advance to the final reaction solution concentration as shown in Table 4 and add to the 96-well microtiter plate prepared as described above, and let it react for a certain period of time. I went there. The initial emission intensity was measured in the same manner as in the method of Example 1. The results are shown in Table 4.
表４に示すとおり、最終反応溶液のｐＨ、ルミノール濃度および過酸化水素濃度を適正に選択して組み合わせることにより、発光反応開始後も高い発光持続性を保つことが確認された。 As shown in Table 4, it was confirmed that high luminescence persistence was maintained even after the start of the luminescence reaction by appropriately selecting and combining the pH, luminol concentration and hydrogen peroxide concentration of the final reaction solution.
市販のＰＯＤをＰＯＤ標識抗体（ＰＯＤ−ＩｇＧ）として用い、化学発光測定に供した場合、最終反応溶液中に含まれるルミノールおよび過酸化水素の濃度範囲が、それぞれ１．０〜３．０ｍＭおよび０．１１〜０．２２ｍＭの範囲内であり、かつ、ルミノール／過酸化水素の濃度比が、４．０〜２０．０において、高い発光安定性および発光持続性が保たれることがわかる。ルミノール／過酸化水素の濃度比が２０．０を越える場合、発光持続性は高く保たれるものの、絶対的な発光強度が低くなってしまい、実測に耐えられないものであった。発光強度の観点から、ルミノール／過酸化水素の濃度比は、４．０〜２０．０が好ましい。 When commercially available POD is used as a POD-labeled antibody (POD-IgG) and subjected to chemiluminescence measurement, the concentration ranges of luminol and hydrogen peroxide contained in the final reaction solution are 1.0 to 3.0 mM and 0, respectively. It can be seen that high luminescence stability and luminescence persistence are maintained when the luminol / hydrogen peroxide concentration ratio is in the range of .11 to 0.22 mM and the concentration ratio is 4.0 to 20.0. When the concentration ratio of luminol / hydrogen peroxide exceeded 20.0, although the luminescence persistence was kept high, the absolute luminescence intensity was lowered, and the measurement was unbearable. From the viewpoint of emission intensity, the concentration ratio of luminol / hydrogen peroxide is preferably 4.0 to 20.0.
［ＰＯＤ標識マウスＩｇＧ抗体を用いた緩衝液の検討］
化学発光反応は、表５に示す各種緩衝液に、ルミノールおよび過酸化水素を溶解し、同じく表５に示すような最終反応液濃度になるように調製した試薬を、実施例３と同様に作製した９６ウェルマイクロタイタープレートに添加し、一定時間反応させることで行った。初発時発光強度は、実施例１と同様に測定した。結果を表５に示す。
[Examination of buffer solution using POD-labeled mouse IgG antibody]
In the chemiluminescence reaction, reagents prepared by dissolving luminol and hydrogen peroxide in various buffer solutions shown in Table 5 and having final reaction solution concentrations as shown in Table 5 were prepared in the same manner as in Example 3. The sample was added to the prepared 96-well microtiter plate and allowed to react for a certain time. The initial emission intensity was measured in the same manner as in Example 1. The results are shown in Table 5.
表５に示すとおり、緩衝液の違いによらず、最終反応溶液のｐＨ、ルミノール濃度および過酸化水素濃度を適正に選択して組み合わせることにより、発光反応開始後も高い発光持続性を保つことが確認された。 As shown in Table 5, it is possible to maintain high luminescence persistence even after the start of the luminescence reaction by properly selecting and combining the pH, luminol concentration and hydrogen peroxide concentration of the final reaction solution, regardless of the buffer solution. confirmed.
Claims (4)
（ｂ）酸化剤
（ｃ）化学発光物質
（ｄ）緩衝液
を含んでなるペルオキシダーゼ化学発光測定試薬であって、化学発光反応開始時において、（ｂ）濃度０．０５〜１．０ｍＭ、（ｃ）濃度０．５〜５．０ｍＭでかつ（ｄ）ｐＨが７．５〜９．５であり、
（ａ）が遊離状態のペルオキシダーゼである場合、（ｂ）および（ｃ）の濃度比が、（ｂ）濃度１に対して、（ｃ）濃度が１．０〜１０．０の範囲で、かつ、（ｄ）のｐＨが７．５〜９．５の範囲であり、
（ａ）が抗体に標識したペルオキシダーゼである場合、（ｂ）および（ｃ）の濃度比が、（ｂ）濃度１に対して、（ｃ）濃度が４．０〜２０．０の範囲で、かつ、（ｄ）ｐＨが８．０〜９．５の範囲であることを特徴とするペルオキシダーゼ化学発光測定試薬。 (A) Basidiomycete-derived peroxidase (b) Oxidizing agent (c) Chemiluminescent substance (d) A peroxidase chemiluminescence measuring reagent comprising a buffer solution. -1.0 mM, (c) concentration 0.5-5.0 mM and (d) pH 7.5-9.5,
When (a) is a free peroxidase, the concentration ratio of (b) and (c) is (c) in the range of 1.0 to 10.0 with respect to (b) concentration 1, and , (D) has a pH in the range of 7.5 to 9.5,
When (a) is a peroxidase labeled with an antibody, the concentration ratio of (b) and (c) is in the range of (c) concentration of 4.0 to 20.0 with respect to (b) concentration 1, (D) A reagent for measuring peroxidase chemiluminescence, wherein the pH is in the range of 8.0 to 9.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2009134576A JP5334691B2 (en) | 2009-06-04 | 2009-06-04 | Peroxidase chemiluminescence reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2009134576A JP5334691B2 (en) | 2009-06-04 | 2009-06-04 | Peroxidase chemiluminescence reagent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2010281652A JP2010281652A (en) | 2010-12-16 |
| JP5334691B2 true JP5334691B2 (en) | 2013-11-06 |
Family
ID=43538523
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2009134576A Expired - Fee Related JP5334691B2 (en) | 2009-06-04 | 2009-06-04 | Peroxidase chemiluminescence reagent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5334691B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105628682B (en) * | 2016-01-04 | 2018-04-06 | 广西科技大学 | Utilize the method for matrine content in composite modified dose of load pyrolytic graphite electrode electrogenerated chemiluminescence behavior determination medicine |
| CN105675583B (en) * | 2016-01-04 | 2018-04-06 | 广西科技大学 | Utilize the method for verapamil hydrochloride content in composite modified dose of load pyrolytic graphite electrode electrogenerated chemiluminescence behavior determination medicine |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6253390A (en) * | 1985-09-02 | 1987-03-09 | Toshiba Corp | Fluorescent substance |
| JPS6279781A (en) * | 1985-10-01 | 1987-04-13 | Takara Shuzo Co Ltd | Production of peroxidase |
| JP2528457B2 (en) * | 1987-03-09 | 1996-08-28 | サントリー株式会社 | Hydrogen peroxide determination method |
| JPH031949A (en) * | 1989-05-30 | 1991-01-08 | Iwasaki Electric Co Ltd | UV curing irradiator |
| JP3005238B2 (en) * | 1990-03-30 | 2000-01-31 | サントリー株式会社 | Chemiluminescence assay |
| JPH0753525A (en) * | 1993-08-10 | 1995-02-28 | Nitto Boseki Co Ltd | Sensitive method for quantifying phosphomonoesterase by chemiluminescence |
| JP3524401B2 (en) * | 1998-09-16 | 2004-05-10 | 株式会社ニチレイ | Enzyme-antibody complex and method for producing the same |
-
2009
- 2009-06-04 JP JP2009134576A patent/JP5334691B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2010281652A (en) | 2010-12-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| del Rıo et al. | Nitric oxide and nitric oxide synthase activity in plants | |
| JP5524865B2 (en) | Method for measuring mevalonic acid, 3-hydroxymethylglutaryl coenzyme A and coenzyme A, and measuring reagent | |
| US9040252B2 (en) | Method for increasing and regulating light emission from a chemiluminescent reaction | |
| Röhlen et al. | Development of an amperometric biosensor platform for the combined determination of l-malic, fumaric, and l-aspartic acid | |
| NZ268405A (en) | Atp-adp chemiluminescent testing for microorganisms including a source of a magnesium ion | |
| Meijer et al. | Overexpression of isocitrate lyase—glyoxylate bypass influence on metabolism in Aspergillus niger | |
| JP5334691B2 (en) | Peroxidase chemiluminescence reagent | |
| JP5311662B2 (en) | Luminescence enhancement method for peroxidase chemiluminescence reaction and luminescence enhancer for peroxidase chemiluminescence reaction | |
| CN101889095A (en) | Luciferase assay methods, reagents and kits | |
| Thompson et al. | 8-Anilino naphthalene sulfonate binding as a probe for conformational changes induced in glutamate dehydrogenase by regulatory reagents | |
| JP2012183018A (en) | L-tryptophane determination method using nad+ dependent type dehydrogenase and kit used for the same | |
| JP5043081B2 (en) | Quantitative determination of L-lysine in biological samples | |
| US7585400B2 (en) | Chip for electrochemical immunoassay | |
| Tang et al. | Flavin specificity and subunit interaction of Vibrio fischeri general NAD (P) H-flavin oxidoreductase FRG/FRase I | |
| Amenta et al. | Nitric oxide in Azospirillum and related bacteria: production and effects | |
| JPH02234698A (en) | Detecting method of dna probe by bacterium-luciferase system | |
| JP2008206491A (en) | METHOD FOR STABILIZING p-HYDROXYBENZOATE HYDROXYLASE | |
| Kim et al. | A commensal symbiotic interrelationship for the growth of Symbiobacterium toebii with its partner bacterium, Geobacillus toebii | |
| KR100821457B1 (en) | Cell line transformed with IDH3A promoter and luciferase-containing vector, and method for screening for obesity inhibition candidate using same | |
| JPS5852640B2 (en) | Method for measuring peroxidase activity | |
| Hamner | The Purification and Thermal Stability of the Peroxidase Enzyme in Cucurbita moschata | |
| JPH05176797A (en) | Method for determining cholesterol and reagent for determination | |
| CN1800852B (en) | An improved rate method for detecting clinical diagnostic reagents | |
| US20090029401A1 (en) | Method for detecting biofuel producing microbes | |
| JPWO2018012606A1 (en) | Reaction accelerator |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20120313 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20120417 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20130207 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20130213 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20130415 |
|
| RD03 | Notification of appointment of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7423 Effective date: 20130415 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20130626 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20130702 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20130724 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20130730 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |