JP2547264C - - Google Patents
Info
- Publication number
- JP2547264C JP2547264C JP2547264C JP 2547264 C JP2547264 C JP 2547264C JP 2547264 C JP2547264 C JP 2547264C
- Authority
- JP
- Japan
- Prior art keywords
- factor
- polyglycoside
- sample
- inhibitor
- activation
- 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
- 239000003112 inhibitor Substances 0.000 claims description 76
- 230000004913 activation Effects 0.000 claims description 62
- 239000002158 endotoxin Substances 0.000 claims description 41
- 229920002498 Beta-glucan Polymers 0.000 claims description 38
- 238000000034 method Methods 0.000 claims description 34
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 30
- 239000003153 chemical reaction reagent Substances 0.000 claims description 28
- 238000012360 testing method Methods 0.000 claims description 27
- 239000006166 lysate Substances 0.000 claims description 24
- 241000239218 Limulus Species 0.000 claims description 23
- 241001529572 Chaceon affinis Species 0.000 claims description 20
- 229920002558 Curdlan Polymers 0.000 claims description 20
- 239000001879 Curdlan Substances 0.000 claims description 20
- 235000019316 curdlan Nutrition 0.000 claims description 20
- 229940078035 curdlan Drugs 0.000 claims description 20
- 239000012190 activator Substances 0.000 claims description 14
- 230000002401 inhibitory effect Effects 0.000 claims description 14
- 239000000872 buffer Substances 0.000 claims description 12
- 239000000758 substrate Substances 0.000 claims description 12
- 239000002243 precursor Substances 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 108010065152 Coagulase Proteins 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims 2
- 239000000523 sample Substances 0.000 description 73
- 238000002360 preparation method Methods 0.000 description 57
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 42
- 239000000243 solution Substances 0.000 description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 29
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 26
- 238000005194 fractionation Methods 0.000 description 20
- 238000005227 gel permeation chromatography Methods 0.000 description 17
- 239000012153 distilled water Substances 0.000 description 16
- 239000000047 product Substances 0.000 description 16
- 239000000126 substance Substances 0.000 description 15
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 14
- 229920001503 Glucan Polymers 0.000 description 11
- DBTMGCOVALSLOR-VPNXCSTESA-N laminarin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1O[C@@H]1[C@@H](O)C(O[C@H]2[C@@H]([C@@H](CO)OC(O)[C@@H]2O)O)O[C@H](CO)[C@H]1O DBTMGCOVALSLOR-VPNXCSTESA-N 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 230000009257 reactivity Effects 0.000 description 11
- 238000006467 substitution reaction Methods 0.000 description 11
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 10
- 150000008131 glucosides Chemical group 0.000 description 10
- 229920001543 Laminarin Polymers 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000008103 glucose Substances 0.000 description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 239000002253 acid Substances 0.000 description 8
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- 238000005345 coagulation Methods 0.000 description 8
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- 238000005259 measurement Methods 0.000 description 8
- 239000012528 membrane Substances 0.000 description 8
- 239000002808 molecular sieve Substances 0.000 description 8
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 8
- 238000012454 limulus amebocyte lysate test Methods 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 6
- 238000001879 gelation Methods 0.000 description 6
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 241000209219 Hordeum Species 0.000 description 5
- 235000007340 Hordeum vulgare Nutrition 0.000 description 5
- 238000000502 dialysis Methods 0.000 description 5
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- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 5
- WZFUQSJFWNHZHM-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)CC(=O)N1CC2=C(CC1)NN=N2 WZFUQSJFWNHZHM-UHFFFAOYSA-N 0.000 description 4
- 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 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 4
- 239000005695 Ammonium acetate Substances 0.000 description 4
- 206010017533 Fungal infection Diseases 0.000 description 4
- 241001466453 Laminaria Species 0.000 description 4
- 208000031888 Mycoses Diseases 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 4
- -1 amine salts Chemical class 0.000 description 4
- 235000019257 ammonium acetate Nutrition 0.000 description 4
- 229940043376 ammonium acetate Drugs 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000000691 measurement method Methods 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 241001474374 Blennius Species 0.000 description 3
- 208000037487 Endotoxemia Diseases 0.000 description 3
- 241000199919 Phaeophyceae Species 0.000 description 3
- 241000239224 Tachypleus tridentatus Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 3
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
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- 230000002538 fungal effect Effects 0.000 description 3
- 229930182478 glucoside Natural products 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000011895 specific detection Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000004879 turbidimetry Methods 0.000 description 3
- OHVLMTFVQDZYHP-UHFFFAOYSA-N 1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-2-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound N1N=NC=2CN(CCC=21)C(CN1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)=O OHVLMTFVQDZYHP-UHFFFAOYSA-N 0.000 description 2
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 2
- 241000588986 Alcaligenes Species 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 2
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000239219 Carcinoscorpius rotundicauda Species 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 150000008151 D-glucosides Chemical class 0.000 description 2
- 241000230129 Eisenia <Phaeophyceae> Species 0.000 description 2
- 102000010911 Enzyme Precursors Human genes 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
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- 206010040047 Sepsis Diseases 0.000 description 2
- 241000239221 Tachypleus gigas Species 0.000 description 2
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- 230000002378 acidificating effect Effects 0.000 description 2
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- 208000027096 gram-negative bacterial infections Diseases 0.000 description 2
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- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 2
- 239000002198 insoluble material Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 2
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- OLBQXBHLZOAVSV-SPIWUIBRSA-N (2R,3R,4S,5R,6R)-4-[(2S,3R,4S,5R,6R)-4-[(2S,3R,4S,5R,6R)-3,5-Dihydroxy-6-(hydroxymethyl)-4-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-(hydroxymethyl)oxane-2,3,5-triol Chemical compound OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](CO)O[C@@H](O[C@H]3[C@H](O)[C@@H](CO)O[C@@H](O[C@@H]4[C@@H](O)[C@H](O)O[C@H](CO)[C@H]4O)[C@@H]3O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O OLBQXBHLZOAVSV-SPIWUIBRSA-N 0.000 description 1
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Description
【発明の詳細な説明】
技術分野
本発明は或る種の酵素前駆体の活性化を阻害する薬剤に関し、さらに詳しくは
、カブトガニ・アメボサイト・ライセートの凝固系に関与する因子のうち、（１
→３）‐β‐Ｄ‐グルカンとの反応により凝固が開始する系の（１→３）‐β‐
Ｄ‐グルカン感受性因子すなわちＧ因子の活性化を阻害する物質、すなわちＧ因
子活性化阻害剤に関する。
背景技術
カブトガニ・アメボサイト・ライセート（Ｈorseshoe crab Ａmebocyte Ｌy
sate−以下、ＬＡＬと略記することがある）が、１９６４年ＬevinとBangにより
グラム陰性細菌の毒素（エンドトキシン）で直ちに凝固（ゲル化）するという現
象が発見されて［Ｊ.Ｌevin and Ｆ.Ｂ.Ｂang（１９６４）Ｂull．Ｊohns Ｈ
opkins Ｈosp.，１１５、２６５−２７４]以来、ＬＡＬはエンドトキシン(細菌
内毒素)の特異的検出測定法、いわゆる「リムルステスト」（Ｌimulus Ｔest）
試薬として広く利用されている。カブトガニの現存種は３属４種でＬimulus po
lyphemus、Ｔachypleus tridentatus、Ｔ.gigas及びＣarcinoscorpius rotundi
caudaが知られており、中でも北米産のＬ．polyphemus、日本、中国産のＴ．tri
dentatusのアメボサイトライセートを用いる「リムルステスト」試薬の商品化が行
なわれている。［例えば、Ｐrogress in Ｃlinical and Ｂiological Ｒese
arch；volume ９３，Ｅndotoxins and their Ｄetection with the Ｌimu
lus Ａmebocyte Ｌysate Ｔest，Ｓtanly Ｗ．Ｗatson，Ｊack Ｌevin and
Ｔhomas Ｊ.Ｎovitsky，Ｅditors：Ｐublished in １９８２ by Ａlan
Ｒ．Ｌiss，Ｉnc．page７‐２４，Ｔitle：Ｔhe Ｌimulus Ｔest and Ｂact
erial Ｅndotoxins：Ｓome Ｐerspectives by Ｊ．Ｌevin参照］。
ＬＡＬは当初エンドトキシンのみに特異的に反応すると考えられていたが、最
近の研究で、ＬＡＬはエンドトキシンと同様に(１→３)-β-Ｄ-グルカンとも反
応することが判明した。ＬＡＬの凝固系は、哺乳動物の血液凝固系と同様に、複
数の凝固因子の段階的反応による系からなり、エンドトキシンにより反応開始す
る系（Ｃ因子活性化系）の他に、（１→３）‐β‐Ｄ‐グルカンによって反応開
始する系（Ｇ因子活性化系）も存在することが確認されている［Ｔ.Ｍorita et
al．（１９８１）ＦＥＢＳ ＬＥＴＴＥＲＳ、１２９、３１８‐３２１及びＳ.
Ｉwanaga et al．（１９８６）Ｊ.Ｐrotein Ｃhem.，５、２５５‐２６８］。
そのため、リムルステストをできるだけエンドトキシン特異的にするための研究
が行なわれており、例えば、Ｔ.Ｏbayashi et al．（１９８５）Ｃlin．Ｃhim．
Ａcta、１４９、５５‐６５には、ＬＡＬの凝固因子の分画‐再構成によりＧ因
子を分離除去した試薬を用いたエンドトキシンの測定法が提案されており、これ
によるエンドトキシン特異的測定キットが生化学工業（株）より「エンドスペシ
ー」なる商品名で販売されている。
しかし、上記提案の測定法はエンドトキシンに特異的な検出測定法としては極
めて強い要求があるが、複数の凝固系因子からなるカブトガニ・アメボサイト・
ライセートからＧ因子を分離除去することは、エンドトキシンや（１→３）‐
β‐Ｄ‐グルカンの不在下で各因子の分画操作を行う必要があり、このために用
いる器具、装置、薬剤に対するエンドトキシンや（１→３）‐β‐Ｄ‐グルカン
の完全除去を事前に行う煩雑な操作が伴うこと、分画操作に伴いアメボサイト
ライセートの希釈化が起り、必要に応じ濃縮操作を要すること、操作が加わる
毎に各因子の活性低下や画分の損失が伴うこと、Ｇ因子と共にコアギユローゲ
ン
（凝固タンパク前駆体）が分離除去されるため、他の因子の再構成により得られ
る測定法は、「リムルステスト」のうちゲル化現象を利用するゲル化法、比濁法
、比濁時間分析法等には適用できず、合成基質法にのみ適用できるという使用制
限が伴う等の欠点を有する。
本発明者らは、ＬＡＬ凝固機構のうち、（１→３）‐β‐Ｄ‐グルカンの関与
により凝固（ゲル化）反応が開始する系（Ｇ因子活性化系）について詳細に研究
を行なっている過程で、これまでＧ因子の活性化にのみ関与すると考えられた（
１→３）‐β‐Ｄ‐グルカン類のうち、（１→３）−β−Ｄ−グルコシド構造単
位が特定の個数だけ連続して結合している構造部分を含有するものは、全く意外
にも、Ｇ因子の活性化とは全く逆の阻害作用を示すことを見い出し本発明を完成
するに至った。
発明の開示
かくして、本発明によれば、下記式で示される（１→３）‐β‐Ｄ‐グルコシド構造単位（分子量：１６２）が連続
して２〜３７０個結合したポリ−（１→３）−β−Ｄ−グルコシド構造部分を少
なくとも１つ含有するポリグリコシドを有効成分とするカブトガニ・アメボサイ
ト・ライセートＧ因子活性化阻害剤が提供される。
本発明によればまた、上記ポリグリコシドの有効量を、カブトガニ・アメボサ
イト・ライセート（ＬＡＬ）に添加することからなる、ＬＡＬ
中に存在するＧ因子の活性化を阻害する方法が提供される。
以下、本発明についてさらに詳細に説明する。 本発明の阻害剤において有効
成分として使用されるポリグリコシドは、下記式
で示される（１→３）‐β‐Ｄ‐グルコシド構造単位が連続して２〜３７０個、
好ましくは３〜３１０個、より好ましくは４〜１８０個結合したポリ−（１→３
）−β−Ｄ−グルコシド構造部分［以下、便宜上これを「ポリ（１→３）グルコ
シド構造部分」という］を１分子中に少なくとも１つ含有するポリグリコシドで
ある。
かくして、本発明で使用されるポリグリコシドは、ポリ（１→３）グルコシド
構造部分を１分子中に少なくとも１つ含有するものである限り、該ポリグリコシ
ド分子の他の部分の構造は、特に制限はなく広い範囲から選ぶことができる。た
だし、他の構造部分はエンドトキシン及びＣ因子活性化系と実質的に相互作用し
ないものであることが重要である。例えば、本発明で使用されるポリグリコシド
は、実質的に１つのポリ（１→３）グルコシド構造部分のみからなるもの、例え
ば下記式
式中、
ｎは２〜３７０、好ましくは３〜３１０、より好ましくは４〜１８０の整数で
ある、
で示されるポリ−（１→３）−β−Ｄ−グルコシドであることができ、或いは１
つのポリ（１→３）グルコシド構造部分に、下記式
で示される（１→４）‐β‐Ｄ‐グルコシド構造単位の１つもしくはそれ以上及
び／又は下記式で示される（１→６）‐β‐Ｄ‐グルコシド構造単位の１つもしくはそれ以上及
び／又は下記式
式（IV）、（Ｖ）、（VI）中、Ｒ1、Ｒ2及びＲ3の少くとも１つはメチル基
、ヒドロキシメチル基、カルボキシメチル基、アセチル基、硫酸基、リン酸基等
化学的に導入し得る基および塩を形成できるものについてはそれらの金属塩、ア
ンモニウム塩及び有機アミン塩から選ばれる残基を表わし、そして残りは水素原
子を表わす、
で示される修飾されたβ−Ｄ−グルコシド構造単位の１つもしくはそれ以上から
構成される糖鎖が結合した［この糖鎖は前記ポリ（１→３）グルコシド構造部分
に分岐鎖として結合していてもよい］構造のものであつてもよい。
さらにまた、本発明で使用するポリグリコシドは、２つ又はそれ以上の前記ポ
リ（１→３）グルコシド構造部分が、他の糖鎖構造部分を狭んで下記式
Ａ1−Ｂ1−Ａ2−Ｂ2−・・・・・・・
式中、
Ａ1、Ａ2、・・・・・はそれぞれ前記式（Ｉ）で示される（１→３）−β−Ｄ
−グルコシド構造単位が連続して２〜３７０個、好ましくは３〜３１０個、より
好ましくは４〜１８０個結合したポリ−（１→３）−β−Ｄ−グルコシド構造部
分を表わし、Ａ1、Ａ2、・・・・・の各構造部分を構成する式（Ｉ）の単位の数
は互に異なっていてもよく、そしてＢ1、Ｂ2、・・・・・は各々同一もしくは相
異なる
他の糖鎖構造部分を表わす、
で示されるように連結した構造のものであつてもよい。ここで、Ｂ1、Ｂ2、・・
・・・によつて表わされる他の糖鎖構造部分としては、例えば前記式（II）、（
III）、（IV）、（Ｖ）又は（VI）で示される構造単位の１個又は２個以上のブ
ロツクからなる構造部分が挙げられる。
さらにまた、本発明で使用するポリグリコシドは、前記ポリ（１→３）グルコ
シド構造部分と、上記Ｂ1、Ｂ2、・・・・・によつて表わされる如き他の糖鎖構
造を狭んで前記式（Ｉ）で示される（１→３）−β−Ｄ−グルコシド構造単位が
連続して３７１個以上結合した長鎖のポリ−（１→３）−β−Ｄ−グルコシド構
造部分とが連結した構造のものであつてもよい。
従つて、本発明で使用されるポリグリコシドは前記ポリ（１→３）グルコシド
構造部分を１分子中に少なくとも１つ含有するものである限り、その分子量は特
に制約されるものではないが、水に対する溶解度が低下したり、粘度が増大する
ことにより取扱が困難となり、エンドトキシン測定キット調製時の一定品質を得
る点等から不利を生ずることがあるので、一般には分子量が５００，０００以下
、好ましくは５００〜２４０，０００、さらに好ましくは６５０〜８０,０００
の範囲内のものが好都合である。
また、本発明で使用されるポリグリコシドは前記のポリ（１→３）グルコシド
構造部分を１分子中に少なくとも１つ含有するものから実質的になることが好ま
しいが、しかし必ずしもそうである必要はなく、例えば、前記式（Ｉ）で示され
る（１→３）−β−Ｄ−グルコシド構造単位が連続して３７１個以上結合した高
分子量のポリ−（１→３）−β−Ｄ
−グルコシド構造部分を含有する他のポリグリコシドが混在していてもよい。何
んとなれば、本発明によるポリグリコシドは、ＬＡＬのＧ因子活性化系の開始因
子であるＧ因子に、Ｇ因子活性化物質である高分子量のポリ−（１→３）−β−
Ｄ−グルコシドよりも速く強く結合して活性型Ｇ因子への活性化を阻害し、かか
る高分子量のポリ−（１→３）−β−Ｄ−グルコシドの存在によってその阻害作
用に実質的な影響がないからである。
上記の如く他の成分が混在するポリグリコシドを阻害剤として使用する場合、
本発明によるポリグリコシドの阻害剤中における含有量には特に制限はないが、
その含有量があまりにも少ないと、Ｇ因子の活性化の阻害に多量の該ポリグリコ
シドを使用することが必要となり不経済であるので、一般には、少なくとも５重
量％、好ましくは１０重量％以上、さらに好ましくは２０重量％以上を占めるこ
とが望ましい。
なお、本明細書においてポリグリコシドの分子量は、分子量既知の標準物質を
用い下記の条件でゲルパーミエイションクロマトグラフイーを行ない標準曲線を
作成し、次に供試試料について同じ条件でクロマトグラフイーを行ない、その結
果を標準曲線と対比することにより求めた値である。
カラム：TSKgel G-PWXLシリーズ(東ソ-株式会社)7.8×300mm数種数本
移動相：0.3MNaOH
流速：0.5ml／min
試料溶液濃度：0.1-5mg／ml
試料溶液注入量：0.1ml
カラム温度：室温
検出法：示差屈折計(ＬＫＢ社)による測定又はフエノール硫酸法による糖定量
標準物質：TSK 標準ポリエチレンオキシド(東ソー株式会社)およびポリエチレン
グリコール（半井化学薬品株式会社）の重量平均分子量が１,０００から８６０,
０００の１０種を使用。
本発明においてＧ因子活性化阻害剤として用いられる上記の如き特性をもつポ
リグリコシドは、天然に由来するものであってもよく、或いは合成されたもの又
は前記式（Ｉ）で示される（１→３）−β−Ｄ−グルコシド構造単位を３個以上
含有するポリ（１→３）−β−Ｄ−グルコシドの一部を化学的に修飾したもので
あつてもよい。通常は天然に由来するものの方が入手容易である。そのようなポ
リグリコシドの具体例としては以下に記載するものが挙げられる。
(１) 前記式（Ｉ）で示される（１→３）‐β‐Ｄ‐グルコシド構造単位のみか
ら実質的になる実質的に直鎖状のポリグルコシド：例えば、アルカリゲネス属（
Ａlcaligenes）バクテリア由来の（１→３）‐β‐Ｄ‐グルカン；鞭毛藻（Ｅug
lena）由来のパラミロン；高等植物の繊維組織のβ‐グルカン又は篩管から抽出
されるカロース；上記（１→３）−β−Ｄ−グルカンやＬaminaria(コンブ属)、
Ｅisenia（アラメ属）等の褐藻類由来のラミナラン類等の部分水解物中に含まれ
る高重合度の（１→３）‐β‐結合からなるＤ‐グルコース重合体；ラミナリデ
キストリン(重合度１０〜２０のもの)、ラミナリオリゴ糖(重合度１０以下のも
の)、等。
(２) 前記式（Ｉ）で示されるポリ−（１→３）‐β‐Ｄ‐グルコシド構造単位
と前記式（III）で示される（１→６）‐β‐Ｄ‐グルコシド構
造単位の両者を含有するポリグリコシド：例えば、
a）（１→３）‐β‐結合からなる主鎖に１〜数個の（１→６）‐β‐結合で
連鎖したグルコース又はグルコース重合体が組込まれたもの、例えば、Ｅisenia
（アラメ属）褐藻類由来のラミナラン類。
b）上記a）の（１→３）‐β‐結合で連鎖したグルコース又はグルコース重合
体にさらに（１→３）‐β‐結合の糖鎖が（１→６）‐β‐結合で分岐し、また
特に糖鎖の一部に他の糖部分を含みうるもの、例えば、Ｌaminaria（コンブ属）
褐藻類由来のラミナラン類、Ｏchromonas、Ｐhaeodactylum、Ｓkeletonema、Ｂi
ddulphia、Ｃoscinodiscus、Ｃhaetoceros等の珪藻由来のクリソラミナラン類、
Ｐoria（ブクリヨウ菌）由来のパキマン等。
c）さらに多くの分岐をもち、樹状構造を有するもの、例えばＰhytophthoraの
細胞壁由来のグルカン、等。
d）（１→３）‐β‐結合よりなる直鎖状グルカンに（１→６）‐β‐結合で
グルコースが連結しているもの、例えばグルコース単位３残基当り１残基の割合
で分岐のあるＳclerotinia由来のスクレロタン、Ｓchizophyllum（スエヒロタケ
）のシゾフイラン、Ｓclerotium、Ｃorticium、Ｓtromatinia等に由来するスク
レログルカン類等。
また、（１→３）‐β‐結合よりなる直鎖状グルカンのグルコース単位５残基
当り２残基の割合で（１→６）‐β‐結合でグルコースが結合しているもの、例
えば、Ｌentinus（シイタケ）のレンチナン、等。
e）（１→６）‐β‐結合よりなる直鎖状グルカンのグルコースのＣ‐３位か
ら（１→３）‐β‐結合でグルコース鎖が複数分岐しているもの、例えば、Ｓac
charomyces（パン酵母）の細胞壁由来のβ‐グルカン、等。
(３) 前記式（Ｉ）で示される（１→３）‐β‐Ｄ‐グルコシド構造単位と前記
式（II）で示される（１→４）‐β‐Ｄ‐グルコシド構造単位の両者を含有する
ポリグリコシド：例えば、Ｃetraria、Ｕsnea、Ｅvernia等に由来するリヒエナ
ン類、オオムギ胚乳中に含まれるβ‐グルカン等。
以上に述べたポリグリコシドの或る種のものは市販品として入手することがで
き、それらはそのまま本発明の阻害剤として利用することができるが、必要に応
じて、糖類を部分的に分解し及び／又は分別処理に付して、前記式（Ｉ）で示さ
れる（１→３）‐β‐Ｄ‐グルコシド構造単位を前記特定量で含有するポリグリ
コシドに富む画分を調製し、それを本発明の阻害剤に利用してもよい。
かかる糖鎖の部分的分解及び分別処理はそれ自体既知の方法で行なうことがで
きる。例えば、糖鎖の部分分解は酸またはアルカリ、β‐グルカナーゼを用いる
加水分解、加酢分解、音波処理等により行うことができる。また分子量分画は、
アルコール、アセトン、エーテル等の有機溶媒や塩類を用いる分別沈澱法、分子
篩剤や分子篩膜を用いる分画により行うことができる。
また、上記(1)〜(3)に例示した如きポリグリコシドは、糖鎖の一部を、メチル
基のようなアルキル基、ヒドロキシメチル基のようなヒドロキシアルキル基、カ
ルボキシメチル基の如きカルボキシアルキル基、アセチル基、硫酸基、リン酸基
などの酸基、その他の基によつて化学的に修飾されていてもよい。それらはそれ
自体既知の方法でかかる基を導入することによつて調製することができる［例え
ば、(1)安藤、寺山、西沢、山川編、生化学研究法Ｉ、２８４〜３０３（１９６
７）、朝倉書店、(2)Wh
istler，R．L．ed.; Methods in Carbohydrate Chemistry III,１９３〜２６７
，２７１〜３３１(１９６４)，Academic Press等参照]。特に、Ｇ因子活性化作
用をもつ分子量が約６０,０００以上の（１→３）−β−Ｄ−グルカンは部分的
な化学的修飾によつて、そのポリ−（１→３）−β−Ｄ−グルコシド構造部分に
おける前記式（Ｉ）で示される（１→３）−β−Ｄ−グルコシド構造単位の連続
結合数を３７０個以下にすることにより、本発明の阻害剤として使用できるよう
になる。
しかして、本発明において好適に使用しうるポリグリコシドの具体例を示せば
次のとおりである。
・分子量３４２〜１,６３８のラミナリオリゴ糖、
・分子量１,８００〜３,２５８のラミナリデキストリン、
・平均分子量２,０００〜６０,０００の（１→３）−β−Ｄ−グルカン、
・平均分子量３,０００〜２３,０００のラミナラン、
・平均分子量３,０００〜２０,０００のスクレロタン、
・平均分子量５００,０００以下のシゾフイラン、
・平均分子量１,１００,０００以下のレンチナン、
・平均分子量１２,０００以下のパン酵母グルカン水可溶物、
・平均分子量３３,０００以下のリヒエナン、
・平均分子量２００,０００以下の大麦β−グルカン、
・例えばカードランの部分カルボキシメチル化により得られる平均分子量４０,
０００〜２４０,０００の部分カルボキシメチル化（１→３）−β−Ｄ−グルカ
ンおよびその塩（置換度：０.００３〜１.０）、
・平均分子量２３,０００以下の部分カルボキシメチル化ラミナランおよびその
塩（置換度：１.０以下）、
・平均分子量８０,０００以下の部分メチル化（１→３）−β−Ｄ−グルカン（
置換度：０.００３〜１.０）、
・平均分子量２３,０００以下の部分硫酸化ラミナランおよびその塩(置換度：１
０以下)。
以上に述べた本発明に従うポリグリコシドは、後述する実施例において実証さ
れているように、ＬＡＬ中に存在するＧ因子の活性化を強力に阻害する作用を有
しているので、リムルステストにおいてＧ因子活性化系による影響を受けずに検
体中のエンドトキシンを特異的に検出測定するために使用することができる。そ
の使用に際して、Ｇ因子活性化阻害剤としてのポリグリコシドは、通常、リムル
ステストに用いられるＬＡＬに対し、該ＬＡＬ中のＧ因子の活性化を完全に阻止
するのに必要な量以上を検出測定時に添加するか、ＬＡＬに事前に添加する
か、ＬＡＬの抽出調製時に添加することができる。
ここでＬＡＬ中のＧ因子の活性化を完全に阻止するのに必要な阻害剤の量は、
例えば、次のようにして決定することができる。
氷冷下、一定量のＬＡＬに、通常の測定条件下においてＬＡＬを充分に活性化
する一定量のＧ因子活性化物質（エンドトキシンを含有しないもの、またできる
限りＧ因子活性化阻害剤を含まないもの）を加え、これに対して阻害剤（エンド
トキシンを含有しないもの）を濃度を変えて加え、通常のＬＡＬ使用時と同条件
で反応させる。この条件下でＬＡＬの活性化を１００％阻害する阻害剤の濃度を
求める。
次に、上で求めた濃度の阻害剤を上記の一定量のＬＡＬに加え、更にＧ因子活
性化物質の量を変化させて加え、どの濃度で加えてもＬＡＬが活性化されないこ
とを確認する。
上記の操作で一定量のＬＡＬ中のＧ因子の活性化を完全に阻害するのに必要な
阻害剤の量（濃度）を求めることができる。
このようにして求めた各種市販のライセートのＧ因子活性化を完全に阻止する
のに要するＧ因子活性化阻害剤の量を示せば次のとおりである。
（注）＊Ｇ因子活性化阻害剤：調製例４−１で得たカードランギ酸分解物のＧ
ＰＣ分画画分４（表２中、試料Ｎｏ.１４）。
上記表−１に示す阻害剤の必要量から明らかなように、ＬＡＬからなるリムル
ステスト試薬には、本発明によるポリグリコシドをＬＡＬ１ml当り少なくとも５
０ｎｇ、好ましくは１００ｎｇ以上、さらに好ましくは１００〜２３０ｎｇ、最
も好ましくは２３０〜５００ｎｇの範囲内で添加するのが適当である。
以下、本発明のＧ因子活性化阻害物質の調製法、作用機作、該阻害物質を用い
たリムルステスト試薬、キット等についてさらに詳細に説明する。Description: TECHNICAL FIELD The present invention relates to an agent that inhibits the activation of a certain zymogen, and more specifically, among the factors involved in the coagulation system of horseshoe crab amebosite lysate, (1)
→ (3) -β-D-glucan reacts with (1 → 3) -β-
The present invention relates to a D-glucan sensitive factor, ie, a substance that inhibits the activation of factor G, ie, a factor G activation inhibitor. BACKGROUND ART Horseshoe crab Amebocyte Lysate (Horseshoe crab Amebocyte Lysate)
sate-hereinafter may be abbreviated as LAL), but in 1964 Levin and Bang discovered the phenomenon of immediate coagulation (gelation) with a gram-negative bacterial toxin (endotoxin) [J. Levin and FB .Bang (1964) Bull. Johns H
opkins Hosp., 115 , 265-274], LAL has been used as a specific detection and measurement method for endotoxin (bacterial endotoxin), the so-called "Limulus Test".
It is widely used as a reagent. The existing species of horseshoe crab are Limulus po
lyphemus , Tachypleus tridentatus , T. gigas and Carcinoscorpius rotundi
cauda is known, especially L. americans from North America. polyphemus , T. from Japan and China. tri
Commercialization of the "Limulus Test" reagent using dentatus amebosite lysate is underway. [For example, Progress in Clinical and Biological Resources
arch; volume 93, Endotoxins and their Detection with the Limu
lus Amebocyte Lysate Test, Stanly W. Watson, Jack Levin and
Thomas J. Novitsky, Editors: Published in 1982 by Alan
R. Liss, Inc. page7-24, Title: The Limulus Test and Bact
erial Endotoxins: Some Perspectives by J. See Levin]. Although LAL was initially thought to specifically react only with endotoxin, recent studies have shown that LAL reacts with (1 → 3) -β-D-glucan as well as endotoxin. The coagulation system of LAL, like the blood coagulation system of mammals, consists of a system based on a stepwise reaction of a plurality of coagulation factors. In addition to the system initiated by endotoxin (factor C activation system), (1 → 3) ) -Β-D-glucan has been confirmed to exist (T-Morita et al.).
al. (1981) FEBS LETTERS, 129 , 318-321 and S.L.
Iwanaga et al. (1986) J. Protein Chem., 5 , 255-268].
Therefore, studies have been conducted to make the Limulus test as endotoxin-specific as possible. For example, T. Obayashi et al. (1985) Clin. Chim.
Acta, 149 , 55-65 proposes a method for measuring endotoxin using a reagent from which factor G has been separated and removed by fractionation and reconstitution of the coagulation factor of LAL. It is sold by Chemical Industry Co., Ltd. under the trade name "End Species". However, although the above-mentioned proposed measurement method has an extremely strong demand as an endotoxin-specific detection measurement method, horseshoe crab, amebocyte,
Separation and removal of factor G from the lysate requires endotoxin or (1 → 3)-
It is necessary to carry out fractionation of each factor in the absence of β-D-glucan. For this purpose, complete removal of endotoxin and (1 → 3) -β-D-glucan from instruments, devices and drugs used in advance is required. The complicated operation to be performed, the fractionation operation causes dilution of amebosite lysate, and the concentration operation is required if necessary, and the decrease in the activity of each factor and the loss of the fraction with each additional operation , The coagulation protein (coagulation protein precursor) is separated and removed together with the factor G. Therefore, the measurement method obtained by reconstituting the other factors includes a gelation method using a gelation phenomenon, a turbidimetry, It is not applicable to turbidimetric analysis or the like, but has drawbacks such as its use being limited to the synthetic substrate method. The present inventors have studied in detail the LAL coagulation mechanism in which a coagulation (gelation) reaction is initiated by the involvement of (1 → 3) -β-D-glucan (factor G activation system). In the past, it was previously thought to be involved only in the activation of factor G (
Among the (1 → 3) -β-D-glucans, those containing a structural portion in which a specific number of (1 → 3) -β-D-glucoside structural units are continuously bonded are completely unexpected. Also found that they exhibited an inhibitory action completely opposite to the activation of factor G, and completed the present invention. DISCLOSURE OF THE INVENTION Thus, according to the present invention, the following formula At least one poly- (1 → 3) -β-D-glucoside structure unit in which 2 to 370 (1 → 3) -β-D-glucoside structural units (molecular weight: 162) are continuously bonded. A horseshoe crab, amebosite, and lysate factor G activation inhibitor comprising a polyglycoside as an active ingredient is provided. According to the present invention, there is also provided a method for inhibiting the activation of factor G present in LAL, comprising adding an effective amount of the above-mentioned polyglycoside to horseshoe crab amebosite lysate (LAL). Hereinafter, the present invention will be described in more detail. The polyglycoside used as an active ingredient in the inhibitor of the present invention has the following formula: 2 to 370 consecutive (1 → 3) -β-D-glucoside structural units represented by
Preferably 3 to 310, more preferably 4 to 180 poly- (1 → 3)
) A polyglycoside containing at least one -β-D-glucoside structure portion [hereinafter referred to as “poly (1 → 3) glucoside structure portion” for convenience] in one molecule. Thus, as long as the polyglycoside used in the present invention contains at least one poly (1 → 3) glucoside structure moiety in one molecule, the structure of the other part of the polyglycoside molecule is particularly limited. But you can choose from a wide range. It is important, however, that the other structural parts do not substantially interact with the endotoxin and factor C activation system. For example, the polyglycoside used in the present invention is substantially composed of only one poly (1 → 3) glucoside structure portion, for example, the following formula: Wherein n is an integer from 2 to 370, preferably from 3 to 310, more preferably from 4 to 180. It can be a poly- (1 → 3) -β-D-glucoside represented by
Two poly (1 → 3) glucoside structures have the following formula One or more of the (1 → 4) -β-D-glucoside structural units represented by and / or the following formula One or more of the (1 → 6) -β-D-glucoside structural units represented by and / or the following formula In the formulas (IV), (V) and (VI), at least one of R 1 , R 2 and R 3 is a chemical group such as a methyl group, a hydroxymethyl group, a carboxymethyl group, an acetyl group, a sulfate group or a phosphate group. And those capable of forming a group and a salt which can be introduced into the compound, represent a residue selected from metal salts, ammonium salts and organic amine salts thereof, and the remainder represents a hydrogen atom. A sugar chain composed of one or more glucoside structural units is bonded [this sugar chain may be bonded as a branched chain to the poly (1 → 3) glucoside structural portion]. You may. Furthermore, the polyglycoside used in the present invention has the following formula A 1 -B 1 -A 2-in which two or more of the poly (1 → 3) glucoside structure portions narrow other sugar chain structure portions. B 2 - in ......... formula, A 1, A 2, ( 1 → 3) represented by the formula each ..... is (I) -β-D
- 2-370 amino glucoside structural units continuously, preferably 3-310, more preferably bound 4-180 amino poly - represents (1 → 3) -β-D- glucoside structural portion, A 1, The number of units of the formula (I) constituting each structural part of A 2 ,... May be different from each other, and B 1 , B 2 ,. It may have a structure linked to another sugar chain structural part as shown by the symbol. Where B 1 , B 2 , ...
Are represented by, for example, the above formulas (II) and (
And a structural part comprising one or more blocks of the structural unit represented by (III), (IV), (V) or (VI). Furthermore, the polyglycoside used in the present invention is obtained by narrowing the poly (1 → 3) glucoside structure portion and other sugar chain structures represented by the above B 1 , B 2 ,. A long-chain poly- (1 → 3) -β-D-glucoside structural part in which 371 or more (1 → 3) -β-D-glucoside structural units represented by the formula (I) are continuously bonded to each other. It may have a linked structure. Therefore, the molecular weight of the polyglycoside used in the present invention is not particularly limited as long as it contains at least one poly (1 → 3) glucoside structure in one molecule. In general, the molecular weight is 500,000 or less, and preferably the molecular weight is 500,000 or less. 500-240,000, more preferably 650-80,000
Those within the range are convenient. In addition, the polyglycoside used in the present invention is preferably substantially composed of at least one poly (1 → 3) glucoside structure portion in one molecule, but it is not always necessary. For example, for example, high-molecular-weight poly- (1 → 3) -β-D-glucoside in which 371 or more (1 → 3) -β-D-glucoside structural units represented by the formula (I) are continuously bonded. Other polyglycosides containing a structural part may be mixed. What is necessary is that the polyglycoside according to the present invention contains a high-molecular weight poly- (1 → 3) -β-β-factor which is a factor G activator in addition to the factor G which is an initiator of the factor G activation system of LAL.
It binds faster and more strongly than D-glucoside and inhibits activation to activated factor G, and the presence of such high molecular weight poly- (1 → 3) -β-D-glucoside substantially affects its inhibitory action. Because there is no. When using a polyglycoside mixed with other components as an inhibitor as described above,
The content of the polyglycoside according to the present invention in the inhibitor is not particularly limited,
If the content is too low, it is necessary to use a large amount of the polyglycoside to inhibit the activation of factor G, which is uneconomical, so that it is generally at least 5% by weight, preferably 10% by weight or more. More preferably, it accounts for 20% by weight or more. In this specification, the molecular weight of the polyglycoside is determined by performing gel permeation chromatography using a standard substance having a known molecular weight under the following conditions to prepare a standard curve, and then performing chromatography on the test sample under the same conditions. , And comparing the result with a standard curve. Column: TSKgel G-PW XL series (Toso-Co., Ltd.) 7.8 × 300 mm several mobile phases: 0.3 M NaOH Flow rate: 0.5 ml / min Sample solution concentration: 0.1-5 mg / ml Sample solution injection volume: 0.1 ml Column Temperature: room temperature detection method: measurement by differential refractometer (LKB) or sugar determination by phenol sulfuric acid method Standard substance: TSK standard polyethylene oxide (Tosoh Corporation) and polyethylene glycol (Hansui Chemicals) have a weight average molecular weight of 1 000 to 860,
Use 10 kinds of 000. The polyglycoside having the above properties used as the factor G activation inhibitor in the present invention may be derived from nature, or may be synthesized or represented by the above formula (I) (1 → 3) Partially modified poly (1 → 3) -β-D-glucoside containing three or more -β-D-glucoside structural units may be used. Usually, those derived from nature are easier to obtain. Specific examples of such polyglycosides include those described below. (1) A substantially linear polyglucoside substantially consisting only of the (1 → 3) -β-D-glucoside structural unit represented by the formula (I): For example, a genus Alcaligenes (
Alcaligenes ) (1 → 3) -β-D-glucan from bacteria; flagellates ( Eug
lena ) derived paramylon ; β-glucan of higher plant fiber tissue or callose extracted from phloem tubes; (1 → 3) -β-D-glucan or Laminaria
Highly polymerized D-glucose polymer composed of (1 → 3) -β-bonds contained in partially hydrolyzed products such as laminarans derived from brown algae such as Eisenia ; and laminaridextrin (polymerization degree 10 -20), laminari-oligosaccharides (polymerization degree of 10 or less), and the like. (2) Both the poly- (1 → 3) -β-D-glucoside structural unit represented by the formula (I) and the (1 → 6) -β-D-glucoside structural unit represented by the formula (III) Polyglycosides containing: for example, a) glucose or a glucose polymer linked by one or several (1 → 6) -β-linkages into a main chain composed of (1 → 3) -β-linkages Stuff, for example, Eisenia
(Alame) Laminarans derived from brown algae. b) The glucose or glucose polymer linked by the (1 → 3) -β-linkage of the above a) further has a (1 → 3) -β-linked sugar chain branched by the (1 → 6) -β-linkage. And, in particular, those which may contain other sugar moieties in part of the sugar chains, eg Laminaria
Laminarans derived from brown algae, Ochromonas , Phaeodactylum , Skeletonema , Bi
Chrysoraminarans derived from diatoms such as ddulphia , Coscinodiscus , Chaetoceros ,
Pakiman and the like derived from Poria . c) Those having more branches and a dendritic structure, such as glucans derived from the cell wall of Phytophthora . d) A straight-chain glucan consisting of (1 → 3) -β-linkage linked to glucose by (1 → 6) -β-linkage, for example, branched at a ratio of 1 residue per 3 residues of glucose unit. And scleroglucans derived from Sclerotinia derived from Sclerotinia , Schizophyllum of Schizophyllum (Suehirotake mushroom), Sclerotium , Corticium , Stromatinia and the like. Further, a linear glucan composed of (1 → 3) -β-bonds, in which glucose is bound by (1 → 6) -β-linkage at a rate of 2 residues per 5 glucose units of glucose units, for example, Lentinus Lentinus , etc. e) A straight-chain glucan consisting of (1 → 6) -β-bonds in which glucose chains are branched at the C-3 position of glucose at (1 → 3) -β-bonds, for example, Sac
β-glucan derived from the cell wall of charomyces (baker's yeast), etc. (3) Contains both the (1 → 3) -β-D-glucoside structural unit represented by the formula (I) and the (1 → 4) -β-D-glucoside structural unit represented by the formula (II) Polyglycosides: for example, Lihyenans derived from Cetraria , Usnea , Evernia, etc., and β-glucan contained in barley endosperm. Certain of the above-mentioned polyglycosides can be obtained as commercial products, and they can be used as inhibitors as they are in the present invention. And / or subjecting it to a fractionation treatment to prepare a polyglycoside-rich fraction containing the (1 → 3) -β-D-glucoside structural unit represented by the formula (I) in the specific amount, It may be used for the inhibitor of the present invention. Such partial decomposition and fractionation of sugar chains can be performed by a method known per se. For example, partial decomposition of a sugar chain can be performed by hydrolysis using an acid or alkali, β-glucanase, vinegar decomposition, sonication, or the like. The molecular weight fraction is
It can be performed by a fractional precipitation method using an organic solvent such as alcohol, acetone or ether or a salt, or by a fractionation using a molecular sieve or a molecular sieve membrane. Further, the polyglycoside as exemplified in the above (1) to (3), a part of the sugar chain, an alkyl group such as a methyl group, a hydroxyalkyl group such as a hydroxymethyl group, a carboxyalkyl group such as a carboxymethyl group. It may be chemically modified with a group, an acetyl group, an acid group such as a sulfate group or a phosphate group, or another group. They can be prepared by introducing such a group by a method known per se [for example, (1) Ed. Ando, Terayama, Nishizawa, Yamakawa, Biochemical Research Method I, 284-303 (196)
7), Asakura Shoten, (2) Whistler, R. L. ed .; Methods in Carbohydrate Chemistry III, 193-267
271-331 (1964), Academic Press et al.]. In particular, (1 → 3) -β-D-glucan having a molecular weight of about 60,000 or more, which has a factor G activating effect, is partially poly-modified to obtain its poly- (1 → 3) -β-glucan. By making the number of continuous bonds of the (1 → 3) -β-D-glucoside structural unit represented by the formula (I) in the D-glucoside structural portion to be 370 or less, it can be used as the inhibitor of the present invention. Become. The specific examples of polyglycosides that can be suitably used in the present invention are as follows. A laminarioligosaccharide having a molecular weight of 342 to 1,638, a laminaridextrin having a molecular weight of 1,800 to 3,258, a (1 → 3) -β-D-glucan having an average molecular weight of 2,000 to 60,000, and an average. Laminaran having a molecular weight of 3,000 to 23,000, ・ Sclerotan having an average molecular weight of 3,000 to 20,000, ・ Schizofirane having an average molecular weight of 500,000 or less, ・ Lentinan having an average molecular weight of 1,100,000 or less, ・ Average molecular weight 12 Water soluble matter of baker's yeast glucan of not more than 2,000, ・ Lihyenan having an average molecular weight of not more than 33,000, ・ barley β-glucan having an average molecular weight of not more than 200,000, ・ average molecular weight obtained by partial carboxymethylation of curdlan, for example, 40 ,
Partially carboxymethylated (1 → 3) -β-D-glucan of 000 to 240,000 and salts thereof (degree of substitution: 0.003 to 1.0) Partially carboxymethylated laminaran having an average molecular weight of 23,000 or less And a salt thereof (degree of substitution: 1.0 or less), partially methylated (1 → 3) -β-D-glucan having an average molecular weight of 80,000 or less (
Degree of substitution: 0.003 to 1.0) Partially sulfated laminaran having an average molecular weight of 23,000 or less and a salt thereof (degree of substitution: 1)
0 or less). As described above, the polyglycoside according to the present invention has a potent inhibitory effect on the activation of factor G present in LAL, as demonstrated in the examples described below. It can be used to specifically detect and measure endotoxin in a sample without being affected by the activation system. At the time of its use, the amount of polyglycoside as a factor G activation inhibitor is usually higher than the amount required to completely inhibit the activation of factor G in the LAL with respect to LAL used in the Limulus test. It can be added, pre-added to LAL, or added during LAL extraction preparation. Here, the amount of inhibitor required to completely block the activation of factor G in LAL is
For example, it can be determined as follows. Under ice cooling, a certain amount of LAL is added to a certain amount of a factor G activator (which does not contain endotoxin and contains as little G factor activation inhibitor as possible) ), And an inhibitor (one that does not contain endotoxin) is added at a different concentration, and the reaction is carried out under the same conditions as when using normal LAL. Under these conditions, the concentration of the inhibitor that inhibits the activation of LAL by 100% is determined. Next, the above-mentioned concentration of the inhibitor was added to the above-mentioned fixed amount of LAL, and the amount of the factor G activator was further changed to confirm that LAL was not activated at any concentration. . By the above operation, the amount (concentration) of the inhibitor required to completely inhibit the activation of factor G in a certain amount of LAL can be determined. The amounts of the factor G activation inhibitor required to completely inhibit the factor G activation of the various commercially available lysates thus obtained are as follows. (Note) * Factor G activation inhibitor: G of curdlangic acid hydrolyzate obtained in Preparation Example 4-1
PC fraction Fraction 4 (Sample No. 14 in Table 2). As is clear from the required amounts of the inhibitors shown in Table 1 above, the Limulus test reagent consisting of LAL contains the polyglycoside of the present invention in an amount of at least 5 per ml of LAL.
0 ng, preferably 100 ng or more, more preferably 100 to 230 ng, most preferably 230 to 500 ng. Hereinafter, the method for preparing the factor G activation inhibitor of the present invention, the mechanism of action, and the Limulus test reagent, kit, and the like using the inhibitor will be described in more detail.
【図面の簡単な説明】
第１図は市販カードランの分子篩（ＧＰＣ）分画パターンであり、
第２図は第１図中Ｎｏ.４４〜４６画分の再クロマト分画パターンである。
［本発明のＧ因子活性化阻害物質の調製法］
本発明のＧ因子活性化阻害物質は、例えば、以下の調製例に示す方法により調
製できる。また、市販品の（１→３）‐β‐Ｄ‐グルカンのうち、本発明の範囲
にあるものは、そのまま使用することが出来る。
調製例１：市販カードランからの分子篩クロマト分画による調製
カードラン(和光純薬工業、試薬、Ｌot Ｎo.ＰＥＱ ９０８０、Ｍn＞１３６,
０００、Ｍw／Ｍn＞２.７６)試料Ｎo.１０１の１gを０.３ＭＮａＯＨに５mg／ml
の濃度に溶解して、１００μlづつ、室温下、以下の条件下でゲルパーミエイシ
ヨンクロマトグラフイー（以下ＧＰＣ）を行なった。｛カラム：ＴＳＫgelＧ６
０００ＰＷXLとＧ５０００ＰＷXL（ともに７.８×３００mm）とを直列に連結、
移動相：０.３ＭＮａＯＨ、流速：０.５ml／min｝。溶出してきた低分子画分（
Ｎｏ.４４〜４６）を採
取し、再クロマトグラフイーにかけ、数平均分子量が３,０５０、多分散度が１.
２９の試料０.０１５mgを得た（試料Ｎo.１）。上記ＧＰＣ分画パターンを添付
の第１図に示す。更に第１図中Ｎo.４４〜４６画分を再クロマトした分画パター
ンを添付の第２図に示す。
本試料Ｎo.１をβ‐１,３‐グルカナーゼ（ザイモリエイス‐１００Ｔ、生化
学工業製）で消化し、該酵素消化液をＧＰＣ（カラム：ＴＳＫgelＧ４０００Ｐ
ＷXL、Ｇ３０００ＰＷXL、Ｇ２５００ＰＷXL直列；移動相：蒸留水、流速：０.
６ml／min）で分析し、酵素消化液中の糖組成（グルコース４０％、ラミナリビ
オース３０％、ラミナリトリオース２０％、ラミナリテトラオース８％、ラミナ
リペンタオース２％、回収率９４％）が確認出来た。このことから本試料（Ｎo.
１）の糖構造は（１→３）‐β‐Ｄ‐グルコシド構造部分を含有するβ‐ポリグ
ルコシドであることがわかる。
調製例２：カードランの水に対する溶解度差による分画
市販カードラン（試料Ｎo.１０１）５０gを蒸留水に懸濁し、下記のフローシ
ートに示す操作により分画を行った。
調製例３：カードラン水不溶性糖画分のギ酸分解による調製
試料Ｎo.１０２の４５gをＫ.Ｏgawaらの方法[Ｃarbohydr．Ｒes.，２９，３９
７〜４０３(１９７３)］によりギ酸分解を行った。操作内容を下記のフローシー
トに示す。調製例４−１：カードランギ酸分解物水可溶性画分の分子篩による再分画
先に示した調製例３で得た水可溶性画分（試料Ｎo.３）０.１５gを蒸留水３０
mlに溶解しＧＰＣ（カラム：ＴＳＫ gelＧ３０００ＰＷXL×２、
Ｇ２５００ＰＷXL×１、移動相：蒸留水、流速０.５ml／min）により各０.５ml
宛分画採取し、再クロマトにより分子量の異なる６種の試料（Ｎo.１１〜１６）
を得た。
調製例４−２：カードランギ酸分解物水不溶性画分の分子篩による再分画
調製例３で得た水不溶性画分（試料Ｎo.４）の０.２gを４０mlの０.３ＭＮａ
ＯＨ溶液に溶解し、ＧＰＣ（カラム：ＴＳＫ gel Ｇ３０００ＰＷXL×２、Ｇ２
５００ＰＷXL×１、移動相：０.３ＭＮａＯＨ溶液、流速０.５ml／min）を用い
上記調製例４−１と同様の操作にて分画、再クロマトを行い溶出液に０.３ＭＨ
Ｃl溶液を加えて中和し、分子量の異なる２種の試料（Ｎo.１７及び１８）を得
た。
調製例５：カードラン水不溶性画分からの音波処理による試料の調製
試料Ｎo.１０２の１gを約１００mlの５mＭ ＮａＯＨ溶液に懸濁し、氷冷下音
波発生機、ソニケーターTM（大岳製作所、型式５２０２ＰＺＴ、東京）により２
０ＫＨｚ、８０wで１２分間音波処理により低分子化を行った。
処理液を５Ｍ ＮａＯＨを用い、最終０.３Ｍ ＮａＯＨ溶液とし、上記調製例
４−２に準じクロマト分画を行い分子量の異なる８種類の試料（Ｎo.１９〜２２
及び１０３〜１０６）を得た。
調製例６−１：海藻由来の阻害物質の調製（Ｉ）
アラメ（Ｅisenia bicyclis）由来の試料は、Ｔ.ＵsuiらＡgric．Ｂiol．Ｃhe
m．４３、６０３〜６１１（１９７９）の方法に従い市販アラメ乾燥藻体（東京
、吹田商店）１００gを粉砕後、８０％エタノールにより低分子可溶画分を抽出
除去し、残渣から、２％ＣａＣｌ2水溶液を用
いラミナラン画分を抽出する。次いで該抽出液にエタノール９５％を用い終濃度
７５％溶液とし、生じた沈澱を遠沈により集め、エタノール洗浄後、粗ラミナラ
ン試料を得る。該粗試料を蒸留水に再溶解し、陰イオン交換体（ＤＥＡＥ‐トヨ
パール）により夾雑する酸性物質（アルギン酸等）及び色素類を除き、エタノー
ル再沈澱から試料Ｎo.２５を得た。
調製例６−２：海藻由来の阻害物質の調製（II）
マコンブＬaminaria japonica由来の試料はＪ.Ｊ.Ｃonnellら、Ｊ.Ｃhem.Ｓoc
.，３４９４（１９５０）の方法に従い、市販マコンブ乾燥藻体(東京、吹田商店
)１００gを粉砕後、０.０９Ｍ ＨＣｌ溶液にて約３日間静置抽出し、不溶物を濾
別し、濾液を更に１日静置し、生ずる少量の沈澱を遠心分離により除去し、上清
に３倍容のエタノールを加え、約７５％溶液とし、生ずる沈澱を遠沈により集め
、アルコール洗浄、乾燥後水溶性ラミナラン画分（試料Ｎo.２７）を得た。
調製例７−１：真菌由来の阻害物質の調製（Ｉ）
真菌Ｓclerotinia libertiana由来の試料スクレロタンは、北原ら、岐大農報
８、１００〜１０５（１９５７）の方法に従ってＳclerotinialibertianaの菌
核の脱脂乾燥粉末（３０ｇ）を水で充分に抽出して得た残渣を７％ＮaＯＨ溶液
で抽出し、抽出液に１０％ＣuＳＯ4溶液を加えて沈澱させ、これを分別して塩酸
酸性メタノールで洗浄して銅を除き、８０％メタノールで洗浄してＨＣｌを除き
、メタノール、エーテルで洗浄乾燥することを３回繰返して精製し、６ｇの試料
Ｎo.２８を得た。
調製例７−２：真菌由来の阻害物質の調製（II）
真菌Ｓchizophyllum commune：スエヒロタケ由来の試料は、市販シゾフイラン
（科研製薬：商品名ソニフイラン、医薬品：Ｌot Ｎｏ.Ｊ６１
０４０）をＫ.Ｔabataら、Ｃarbohydr.Ｒes.，８９ １２１〜１３５（１９８１
）の方法に従い前記調製例５の操作に準じ、水溶液中１０時間音波処理後、アル
カリ条件下分子篩分画により分子量の異なる３種類の試料（Ｎo２９、３０、３
１）を得た。
調製例７−３：真菌由来の阻害物質の調製（III）
酵母Ｓaccharomyces cerevisiae：パン酵母由来のβ‐グルカン試料は、市販
パン酵母グルカン（シグマ社Ｌot Ｎo.５６Ｆ‐４０２７）９０mgに蒸留水５０m
lを加え、室温で２時間撹拌後、遠心分離し、上清約５０mlを、減圧濃縮により
１mlとし不溶物を再度遠心除去し、上清から０.６４mgの試料（Ｎo.３３）を得
た。
調製例８：大麦β‐グルカン由来の試料の調製
市販大麦β‐グルカン（シグマ社、Ｌot Ｎo.５６Ｆ‐０６５２）を０.３Ｍ
ＮａＯＨにより５mg／mlの溶液とし、前記調製例４−２に準じアルカリ条件下、
分子篩分画により分子量分布の狭いβ‐グルカン試料（Ｎo.３６）を得た。
また、上記市販の大麦β−グルカンを５mg／mlの濃度にて熱水に溶解し、その
遠心（３,５００rpm、１０分）上清を前記調製例４−１に準じて蒸留水を移動相
として１００μｌづつ５０回ＧＰＣ分画採取し、更に同条件下にて再分画採取し
て分子量の異なる２種の試料(試料Ｎo.３７、３８)を得た。
調製例９：部分カルボキシメチル化(１→３)−β−Ｄ−グルカン（置換度ＤＳ＝
０．６３）の調製
調製例２に準じて得たカードラン水不溶物をA．E．Clarke and B．A．Stone:P
hytochemistry １，１７５〜１８８（１９６２）の方法に従つ
て、カルボキシメチル化した。即ち、１００ｇのカードラン水不溶物を窒素気流
下０℃で１ｌの５Ｍ ＮａＯＨ溶液に溶解し、これを撹拌しながら２３６ｇのモ
ノクロル酢酸を２００mlの水に溶解したものを滴下して加え、添加後、６０〜６
５℃で２時間撹拌した。生ずるゲルを２.５倍容のエタノール中で強く撹拌し細
粉化し濾過した。７０％のエタノールで充分洗滌してからエタノール、エーテル
、エーテルで洗滌し乾燥した。このものを水７ｌに溶解し、１Ｍ酢酸で中和し、
活性炭４０ｇを加え、室温で１時間撹拌し、濾過した。濾液を減圧濃縮して１ｌ
とし、３倍容のエタノールを加えて沈澱とし、エタノール、エーテルで洗滌し、
濃硫酸上減圧乾燥し、１１３.８５ｇを得た。
得られたカードラン部分カルボキシメチル化(１→３)−β−Ｄ−グルカンは、
D．F．Dursoの硝酸ウラニル法Methods in Carbobydrate Chem.VIII,１２７−１
２９（１９８０）参照に従って測定するとエーテル化度(置換度：Degree of Sub
stitution:DS)は０.６３であつた。これは糖鎖を形成しているグルコース残基１
個当りの置換し得る水酸基３箇のうちの０.６３個が置換されたことを意味する
ものである。
得られた部分カルボキシメチル化（１→３）−β−Ｄ−グルカンの２５ｍｇを
５mlの０．１Ｍ酢酸アンモニウム水溶液に溶解し、ＧＰＣ（カラム：トヨパール
ＨＷ６５Ｆ、５×１００ｃｍ；移動相：０.１Ｍ酢酸アンモニウム水溶液；流速
：５.８ml／min）により分画採取し、別のカラムを用いたＧＰＣ（カラム：ＴＳ
ＫgelＧ６０００ＰＷXL＋Ｇ５０００ＰＷXL＋Ｇ３０００ＰＷXLを直列に使用；
移動相：０.１Ｍ酢酸アンモニウム水溶液；流速：０.６ml／min）により再分画
採取し、分子量分布の狭い試料Ｎo.４１（Ｍｎ＝２３１,０００）を得た。
また、部分カルボキシメチル化（１→３）−β−Ｄ−グルカンの０.３ｇを蒸
留水３０mlに溶解し、音波処理（９ｋＨｚ、１８０〜１３０Ｗ、１時間、音波発
生機として久保田製作所、Insonator Model ２０１Ｍを使用）により低分子化し
た後、そのうち４.５mlに０.５mlの１Ｍ酢酸アンモニウム水溶液を加えて混和後
、上記の試料Ｎｏ.４１を得るための操作と同様な操作にて、ＧＰＣ分画採取お
よびＧＰＣ再分画採取を行い、分子量の異なる２種の試料（Ｎｏ．３９、４０）
を得た。
調製例１０：置換度１.２の部分カルボキシメチル化（１→３）−β−Ｄ−グル
カンの調製
調製例９によつて得られた置換度（ＤＳ）０.６３のカルボキシメチル化（１
→３）−β−Ｄ−グルカン１０ｇを窒素気流下０℃で２５mlの１０.５Ｍ Ｎａ
ＯＨに加えてペーストとし、よく撹拌しながら、それにモノクロル酢酸水溶液（
１０ｇ／１２ml）を加え、６０℃に加温し、４時間撹拌し、冷却してから２Ｍ
ＨＣｌ３０mlを加え、次いで２００mlの塩酸酸性エタノール（４０ml ＨＣｌ／
エタノール）中に注いで生ずる沈澱を集め、７０％エタノールで洗滌後、エタノ
ール、エーテルで洗滌し、減圧乾燥し、試料Ｎｏ.１０７の標品を得た。
調製例９に示したＤＳ＝０.６３の部分カルボキシメチル化（１→３）−β−
Ｄ−グルカンと同様の方法で置換度を測定した結果このものはＤＳ＝１.２０で
あつた。
調製例１１：部分カルボキシメチル化ラミナランの調製
部分カルボキシメチル化ラミナランはLaminaria digitataのラミナラン(シグ
マ社Ｌｏｔ Ｎｏ．７７Ｆ−３８８５)を用い、調製例９の部分カルボキシメチ
ル化法と同様、A．E．Clarke and B．A．Stone：Phytoc
hem．１，１７５（１９６２）に記載の方法に準じ調製し、試料Ｎo.４２（ＤＳ
＝０.０６）の標品を得た。
調製例１２：部分メチル化（１→３）−β−Ｄ−グルカンの調製
調製例２に準じて得たカードラン水不溶物３.０ｇをM．Samec，Kolloid-Beihe
fte ５１，３６９(１９４０)の方法に従い、水８０mlに懸濁し、窒素気流下、飽
和苛性ソーダ水溶液１.３５mlを加え、完全に溶解させ、４℃で、こゝにジメチ
ル硫酸６０ｇを徐々に加え、約１時間後、アセトン中に反応液を滴下し、生ずる
沈澱を集め、アセトンで充分洗滌し、濃硫酸上減圧乾燥し、標記調製品（試料Ｎ
ｏ.４３、ＤＳ＝０.１６）の３.１３ｇを得た。
調製例１３：部分硫酸化ラミナランの調製 Laminaria digitata由来のラミナランの硫酸エステル化はピリジン中でピリジ
ン−３酸化硫黄複合体（和光純薬工業、Ｌｏｔ Ｎｏ．ＰＰＬ８８２３）を用い
て次の如く行った。
充分に乾燥したLaminaria digitata由来のラミナラン（シグマ社、Ｌｏｔ Ｎ
ｏ．７７Ｆ−３８８５）０.５ｇを５０mlの脱水ピリジンに溶解し、ピリジン−
３酸化硫黄複合体１ｇを加え、６０℃で１時間反応させ、水１００mlを加え、冷
却し、ＮａＯＨで中和し、あらかじめアルカリ水溶液で充分洗滌してグルカンを
除去した透析膜（スペクトロポア１,０００カツト）を用いて水に対して透析し
た後、濃縮し、２倍容のアセトンを加えて糖成分を沈澱せしめ、アセトンで洗滌
後、濃硫酸上減圧乾燥し、０.３８ｇの標記調製品を得た（試料Ｎｏ.４４、ＤＳ
＝０.１４）。
なお、調製例１２〜１３に示した各標品のメチル基及び硫酸基の置換
度は、下記文献、の方法に従い測定算出した。
落合、津田、阪本；有機定量分析法(微量篇)、南山堂(１９５６)；
Whistler，R．L．ed.，Methods in Carbohydrate Chemistry III，ｐ２２９
〜２３５，２７７〜２８０（１９６４），Academic Press
［市販試料］
下記市販試料は物性を確認後、そのまま又はアルカリ可溶化後中和し、測定に
供した。
グルコース：(和光純薬、ＪＩＳ特級試薬)：試料Ｎo.１０８
ラミナリオリゴ糖：（生化学工業、ピユアー試薬）：試料Ｎo.５〜１０
ラミナラン Ｅisenia araborea由来：(半井化学、試薬)：試料Ｎo.２３
〃 Ｅ.araborea由来：（東京化成、試薬）：試料Ｎo．２４
〃 Ｌaminaria digitata由来：(シグマ社試薬)：試料Ｎo.２６
レンチナン Ｌentinus edodes(シイタケ)由来：(山之内製薬、医薬Ｌot
Ｎo.ＣＫＣ７)：試料Ｎo.３２
リヒエナン Ｃetraria islandica由来：（シグマ社、試薬）：試料Ｎo.
３４
〃 Ｕsnea barbata由来：（シグマ社、試薬）：試料Ｎo.３５実施例１〜４４
上記の各試料の分子量、Ｇ因子活性化阻害力価等の測定結果を下記表−２に示
す。
表中の分子量は前記ＧＰＣにより求めた下式で定義される数平均分子量（Ｍｎ
）で表し、また、分子量分布は、下式で定義される多分散度（Ｍw／Ｍn）で表わ
す。
ただし、Ｈｉはクロマトグラムを時間で等分に多分割したときのｉ番目のピー
ク高さ（試料濃度）を、Ｍiはｉ番目の分子量を表わす。
Ｇ因子活性化阻害力価は下記に示す［Ｇ因子活性化阻害物質の活性力価測定法
］にて測定し、mg当りの単位として示した。
[Ｇ因子活性化阻害物質（以下ＧＩと略記することがある）の活性力価測定法]
反応混合液２００μｌ中には以下のものを含む。
(1)検体(注１)ＧＩ試料又は純水 50μl
[Ｇ因子活性化物質（ＧＡと略記、注２）] 10pg添加又は無添加
(2)カブトガニライセート凝固酵素
前駆体画分(Ａ280＝2.5)(注３) 30μl
(3)カブトガニライセートＧ因子画分
（Ａ280＝0.9）（注３） 20μl
(4)トリス‐ＨＣl緩衝液(pＨ8.0) 20μmole
(5)MgCl2 20μmole
(6)Boc‐Leu‐Gly‐Arg‐pNA（ｔ−ブトキシ
カルボニル−Ｌ−ロイシル−グリシル−Ｌ
−アルギニン−ｐ−ニトロアニリド） 0.13μmole
上記反応液を３７℃で３０分間インキユベートした後、遊離するpＮＡ（パラ
ニトロアニリン）の量を０.０４％亜硝酸ナトリウム（０.４８Ｍ ＨＣl溶液）
、０.３％スルフアミン酸アンモニウム、０.０７％Ｎ‐１-ナフチルエチレンジ
アミン二塩酸塩のそれぞれ０.５mlを順次加え、ジアゾカップリングにより色調
変換し、５４５nmにおける吸光度（Ａ545）量として測定する。
ＧＩ活性は次式により算出する。
この条件下において、ＧＡによるＧ因子の活性化を１００％阻害するＧＩ量を１
００単位とする。
(注１) 検体のうち、水不溶性のものは、０.３Ｍ ＮａＯＨに溶かした後、等容
の０.３Ｍ ＨＣｌを加えて中和して用いる。
(注２) 前記調製例５で調製したカードラン音波処理物のＧＰＣ分画精製標品（
表‐２、Ｎｏ.１０６，分子量２１６,０００）。
(注３) 文献［T．Obayashi e tal.，Clin．Chim．Acta，１４９，５５〜６５（
１９８５）］に従い日本産カブトガニ T.tridentatusから調製した。
上記表‐２の内容から以下のことが明らかである。
（a）Ｇ因子活性化物質として知られる市販カードラン（試料Ｎo.１０１）中に
は、水溶性低分子量のＧ因子活性化阻害物質が混在している
（試料Ｎo.１、２）。
（b）（１→３）−β−Ｄ−グルコシド構造部分から構成されるポリグリコシド
の(１→３)−β−Ｄ−グルコシド構造単位の連結個数が２〜３７０の範囲内にあ
るポリグリコシドは本発明のＧ因子活性化阻害作用を示す。
（c）阻害活性の認められない高分子量β‐グルカン画分（試料Ｎo.１０２）か
ら各種低分子化操作により調製して得られた分子量６０,０００以下の画分がＧ
因子活性化阻害力価を発現した（試料Ｎo.３、４、１１〜２２）。
（d）重合度が１０以下のポリ−（１→３）−β−Ｄ−グルコシド構造部分とポ
リ−（１→４）−β−Ｄ−グルコシド構造部分とがブロツク状に連結している大
麦β−グルカン［Ballanceら，Carbohyd．Res.，６１，１０７〜１１８（１９７
８）参照］（調製例８、試料Ｎｏ.３６、３７、３８）は何れもラミナリテトラ
オースないしラミナリペンタオース相当の活性を示し、グルカン全体の分子量が
６０,０００以下又は以上てあつても、本発明の阻害剤として使用できる。
（e）調製例１０で得た（１→３）−β−Ｄ−グルカンの部分カルボキシメチル
化したもののうち平均置換度が１.０以上の試料（試料Ｎｏ.１０７ ＤＳ＝１.
２）ではＧ因子活性化阻害効果が消失しており、また、試料Ｎｏ．２６として挙
げた高Ｇ因子活性化阻害力価を示す試料を部分カルボキシメチル化（調製例１１
）により（１→３）−β−Ｄ−グルコシド部分の鎖長を短縮した場合（試料Ｎｏ
．４２）及び調製例１３の硫酸エステル修飾により（１→３）−β−Ｄ−グルコ
シド構造部分を短縮した場合、Ｇ因子活性化阻害効果も低減することが併せて確
認され
た。
（f）（１→３）−β−Ｄ−グルコシド構造部分の重合度が３７０以上を有し、
分子量が６０,０００以上のＧ因子活性化効果を示す（１→３）−β−グルカン
であつても、部分メチル化（調製例１２、試料Ｎｏ．４３）、部分カルボキシメ
チル化（調製例９、試料Ｎｏ．４１）により本発明で規定する構造のものとなっ
た場合、Ｇ因子活性化阻害効果を生ぜしめることが確認できた。
［Ｇ因子に対する本発明の阻害物質の作用機作］
カブトガニ血液凝固系のうちＧ因子活性化系は、Ｔ.Ｍorita ｅｔ ａｌ.,Ｆ
ＥＢＳ Ｌｅｔｔｅｒｓ， １２９，３１８〜３２１（１９８１）により報告さ
れたとおり下記チヤートのごとく示される。 本発明の阻害物質が上記凝固系のどの部分を阻害するかを明らかにする目的で
、次の実験を行った。
反応混合液２００μl中には、Ｇ因子活性化阻害物質（ラミナリヘプタオース
、試料番号１０、Ｉと略記）５μg、Ｇ因子活性化物質（カードラン音波処理物
ＧＰＣ分画画分、試料番号１０６、Ａと略記）３pg、文献［Ｔ.Ｏbayashi et al
.，Ｃlin．Ｃhim．Ａcta、１４９、５５−６５（１９８５）］に従いＬＡＬから
調製したＧ因子画分（Ｇと略記）２０
μl、凝固酵素前駆体画分（Ｐと略記）３０μｌ、Ｔris‐ＨＣl緩衝液（ｐＨ８.
０）２０μmole、ＭgＣｌ2 ２０μmole、合成基質Ｂoc‐Ｌeu‐Ｇly‐Ａrg‐ｐ
ＮＡ(Ｓと略記)０.１３μmoleを含む。
各成分の添加順序および加温条件を変えて、Ｇ因子活性化系の活性化阻害の程
度を、それぞれの実験（１〜５）において、阻害剤無添加時をコントロールとし
て測定した結果を以下に示す。 実験１、２、３から明らかなごとく、Ｇ因子（前駆体）に本発明の阻害物質（
Ｉ）を添加した場合、Ｇ因子活性化物質（Ａ）の存在の有無にかかわらず、Ｇ因
子の活性化は１００％阻害される。
一方、実験４、５から明らかなごとくＧ因子が（Ａ）により一旦活性化された
後は阻害物質（Ｉ）を共存せしめても活性型Ｇ因子の阻害は起
らない。
従って本発明の阻害物質（Ｉ）は、Ｇ因子（前駆体）のみに作用する物質とい
える。
尚、ＬＡＬ中に存在するＧ因子の活性化を１００％阻害する量の本発明の阻害
物質（Ｉ）があれば、如何に活性化物質（Ａ）の量を多量存在せしめても、Ｇ因
子の活性化は生じない。しかし、Ｇ因子を１００％阻害し得ない少量の阻害物質
（Ｉ）の存在下に活性化物質（Ａ）が多量存在せしめた場合は、阻害物質（Ｉ）
により阻害されなかったＧ因子が活性化物質（Ａ）により活性化されることも確
認出来た。
このことにより、Ａ.Ｋakinumaらが、Ｂiochem.Ｂiophys.Ｒes.Ｃommun.，１
０１，４３４〜４３９（１９８１）に指摘した、（１→３）‐β‐Ｄ‐グルカン
誘導体や、Ｔ.ＭoritaらがＰrog．Ｃhim．Ｂiol.Ｒes.，１８９、５３〜６４（
１９８５）に示した各種β‐グルカンによるカブトガニＧ因子活性化能における
最大活性化濃度の存在を解析出来た。
実施例４５：Ｇ因子活性化阻害物質添加、非添加キットのエンドトキシン特異的
測定能比較
本発明のＧ因子活性化阻害物質をＬＡＬ‐Ｔestに添加した場合及び添加しな
かった場合のエンドトキシン特異性比較を次の操作により、各種検体を用いて行
った。
用いたＬＡＬ‐Ｔestの商品は、次の構成からなるトキシカラーテストTM（比
色法、生化学工業製）である。
過塩素酸、水酸化ナトリウム、緩衝液、ライセート＋発色合成基質、
エンドトキシンフリー蒸留水、６エンドトキシン標準品、
（１‐ナフチル）エチレンジアミン二塩酸塩。
上記キット構成のうち、緩衝液にＧ因子活性化阻害物質として試料Ｎo.１３
を５μg／mlの濃度で溶解した溶液を用いてのＬＡＬ反応主剤を溶解した反応
液群（Ａ‐キット）及び該阻害物質を含まない緩衝液を用いてを溶解した反
応液群（Ｂ‐キット）として、各種検体に対するＡ、Ｂ両キットの反応性を比較
し、下記表−３に示す。
注１：Escherichia coli 0111:B4 由来エンドトキシン(デイフコ社)
注２：カードラン水不溶性画分；分子量159,000以上；表２、Ｎo.102
注３：再生セルロース膜であるキユプラアンモニウムレーヨン膜で製造されたホ
ローフアイバー型血液透析器ＡＭ‐Ｎeo‐３,０００（旭メデイカル（株）製）
に蒸留水を灌流させ洗浄した洗液。糖含量はフエノール硫酸法で測定した。
注４：健常者２５名の平均±標準偏差
検体b〜e：敗血症の合併が疑われ、血液培養により、Escherichia coliが検出さ
れた
f、g：同上で、Pseudomonas aeruginosaが検出された
i：同上で、Candida albicansが検出された
j：同上で、Candida guilliermondiiが検出された
h：肺アスペルギルス症
k、l：剖検時に全身性真菌感染症と診断された例
m〜r：再生セルロース膜であるキユプラアンモニウムレーヨン膜で製造され
たホローフアイバー型血液透析器により透析を受けた慢性腎不全患者(微生物に
よる感染は無し)
検体Ｎo.１〜５は、トキシカラーテストの測定マニユアルに従い検体を溶媒に
そのまま溶解し、その０.１mlを試料として反応を行い、また検体Ｎo.６（a〜r
）は、Ｔ.Ｏbayashiの方法[Ｊ.Ｌab．Ｃlin．Ｍed.，１０４，３２１〜３３０（
１９８４）］に従って、上記キット構成、を用い血漿検体を前処理した後そ
の０.１mlを試料として反応に供した。
Ａ、Ｂ両キットの反応性は、各検体を、キット構成、を用いて調製した反
応液に加え、３７℃、３０分間反応せしめ、生ずるpＮＡを 尚、本キット組成の場合の最大反応性は△Ａ545＝１.５であった。
表−３から判るように、本発明のＧ因子活性化阻害物質を含むＡ‐キットなら
びに従来品Ｂ‐キットともに、エンドトキシン検体（Ｎｏ.１）に対し、同一の
反応性を示す結果であったが、Ｇ因子活性化物質であるカードラン水不溶性画分
（検体Ｎo.２）は、Ｂ‐キットにおいて、極めて高度の反応性を示した。一方、
Ａ‐キットにおいて該検体は反応性をまったく示さなかったものの、エンドトキ
シンと併せた場合（検体Ｎo.３）は検体Ｎo.１のエンドトキシンと同一の反応性
を示した。
リムルステスト陽性物質（非エンドトキシン性）として知られているセルロー
ス系透析膜水洗液［Ｆ.Ｃ.Ｐearson et al.，Ａrtif．Ｏrgans，８，２９１〜２
９８（１９８４）］を検体（Ｎo.４、Ｎo.５）として用いた場合の結果も、Ａ‐
キット、Ｂ‐キットともに上記カードラン水不溶性画分および／またはエンドト
キシン添加の場合の挙動と同様の結果が示された。
以上の結果により、本発明のＧ因子活性化阻害剤をＬＡＬと併用する
ことは、エンドトキシン特異的測定を可能とすることが判る。
更に従来のＬＡＬ‐Ｔestで、真のエンドトキセミアか否か、明確に判定出来
ないとされた臨床血液検体について、本発明によるＡ‐キットと従来品のＢ‐キ
ットの比較により、菌培養の結果、グラム陰性菌の存在が確認された検体（Ｎo.
６；b〜g）においてはＡ、Ｂ両キットともに高い反応性が示されたが、一方、（
１→３）‐β‐Ｄ‐グルカンを菌体細胞壁に有することが知られている真菌の存
在が確認された検体（同：h〜l）においては、Ａ‐キットでの反応性が示されず
、Ｂ‐キットに高い反応性が示された。また、臨床症状からはエンドトキセミア
とは見なされない血液透析患者（慢性腎不全）の検体（同：m〜r）においてもＡ
‐キットでの反応性は見られず、Ｂ‐キットでの異常高値は透析膜由来の（１→
３）‐β‐Ｄ‐グルカンによるものであることが予測された。
ＬＡＬ‐Ｔestと本発明のＧ因子活性化阻害剤との組合せにより、上記検体の
ごときエンドトキシンの存在の有無が明確でない感染症、敗血症を疑われている
臨床検体を測定することは、真のグラム陰性菌感染症（エンドトキセミア）を的
確に判別出来る利点に加えるに、真菌感染症を判別出来ることから、感染菌タイ
プの早期判定による該感染菌への適切な治療薬剤の選択、処置、並びにその治療
効果の解析を可能とし、本発明の阻害剤を含むキットの提供は診断、医療等医学
の進歩に多大の寄与が期待出来る。
以下に参考例として、本発明のＧ因子活性化阻害物質とカブトガニ・アメボサ
イト・ライセートの組合せによるエンドトキシン特異的検出測定用キットの調製
例を示す。
参考例１：Ｇ因子活性化阻害剤を市販あるいは既存リムルステスト試薬 に、そのエンドトキシン測定時に添加することにより、エンドトキシン特異的測
定キットとする方法
１−１．リムルステスト試薬（凍結乾燥品）は常法通り、指定された溶解液（
蒸留水または緩衝液）で溶解し、これに本発明のＧ因子活性化阻害剤を検体と同
時あるいは別々に添加する（添加順は問わない）ことによって目的を達成する方
法：
例えば、「プレゲルＳ」(凍結乾燥品；ゲル化法リムルステスト製品；生化学工
業(株))に蒸留水０.１mlを加えて溶解し、該阻害剤（カードランのギ酸分解物の
ＧＰＣ分画画分４；表２、Ｎo.１４）の水溶液（１.２μg／ml）０.０１ml（１
２０μｇ／mlＬＡＬ）と検体０.１mlとを加えて静かに振り混ぜ、３７℃で６０
分間静置加温する時、エンドトキシンのみと反応し、ゲルを形成する。
１-２.リムルステスト試薬溶解液にあらかじめ該阻害剤を溶解し、これを用い
てリムルステスト試薬を溶解することによって目的を達する方法：
例えば、「コーテスト・エンドトキシン」（合成基質法リムルステスト製品：
カービービトラム社）を使用する場合は、ＬＡＬ（凍結乾燥品）１バイアルを、
あらかじめ該阻害剤（ラミナラン；表２中、Ｎｏ.２６）０.７μgを溶かした蒸
留水１.４mlで溶解する。その０.１ml（５００ng／ml ＬＡＬ）に検体０.１mlを
加えて３７℃、１０分間加温し、合成基質（Ｓ‐２４２３）含有緩衝液０.２ml
を加えて３７℃、３分間加温する時、エンドトキシンのみと反応し、黄色を呈す
る。定量に際しては、５０％酢酸溶液を２００μl添加し、４０５nmで吸光度を
測定する。
参考例２：Ｇ因子活性化阻害剤をＬＡＬに事前に添加することにより、 エンドトキシン特異的リムルステスト試薬キットとする方法
２−１．市販のＬＡＬ（いわゆるゲル化法リムルステスト試薬）に事前に該阻
害剤を添加することによって、目的を達する方法：
例えば、「リムルスＨＳ‐テストワコー」（凍結乾燥品；ゲル化法リムルステ
スト製品；和光純薬工業（株））を使用して比濁時間分析法により測定する場合
は、ＬＡＬ（凍結乾燥品）１バイアルを、あらかじめ該阻害剤（カードランギ酸
分解物のＧＰＣ分画画分４；表２中、Ｎo.１４）０.５μgを溶かした蒸留水５ml
で溶解する。その０.１ml（１００ｎｇ／mlＬＡＬ）を反応用試験管に分注し、
さらに検体０.１mlを加えて、静かに振り混ぜ、比濁時間分析装置「トキシノメ
ーターＥＴ−２０１」（和光純薬工業）の専用アナリシスモジユール（３７℃）
の所定の測光位置にセットし、スタートスイッチを入れる。エンドトキシンのみ
が反応し、ゲル化時間が表示される。
２−２．あらかじめ調製したＬＡＬに検体添加前に該阻害物質を添加すること
によって目的を達する方法：
例えば、ＬＡＬを用いて比濁法により測定する場合は、カブトガニ（Ｔachypl
eus tridentatus）の血球から低張緩衝液を用いて抽出されたライセート０.１m
lに該阻害剤(ラミナラン；表２中、Ｎo.２６)を１ml当り０.５μｇ溶かした１Ｍ
Ｔris‐ＨＣl‐１Ｍ ＭgＣl2緩衝液（pＨ８.０）溶液０.０１ml（５０ｎｇ／m
lＬＡＬ）を加える。これに検体０.１mlを加えて、３７℃で加温する。エンドト
キシンのみが反応し、白濁する。経時的に６６０nmで吸光度を測定すれば定量で
きる。
参考例３：Ｇ因子活性化阻害剤をＬＡＬの抽出調製時に添加することにより、エ
ンドトキシン特異的リムルステスト試薬原料ライセートを製造 する方法
たとえば、カブトガニ（Ｔachypleus tridentatus、Ｔ. gigas、Ｌimulus pol
yphemus、Ｃarcinoscorpius rotundicauda のどれでもよい）の血リンパ液を
採取し、遠心分離して血球（約２０g）を得、これに２.０mg／lの該阻害剤（部
分カルボキシメチル化ラミナラン；表２中、Ｎｏ.４２）を蒸留水または０.０２
Ｍ Ｔris‐HCl 緩衝液（pＨ８.０）のような低張緩衝液に溶解した該阻害剤溶
液１００mlを加えて、ワーリング・ブレンダーでホモゲナイズした後、遠心分離
（８,０００rpm；３０分間；４℃）により上清と沈澱物に分画する。この操作を
もう一度繰り返し、上清合計約１５０mlをＬＡＬとして得る。このＬＡＬの一定
量を従来法で抽出されたＬＡＬの代わりに用いてリムルステスト試薬（ゲル化法
、比濁法、比濁時間分析法、合成基質法）を調製するとき、エンドトキシンにの
み特異的に反応するエンドトキシン特異的リムルステスト試薬を製造することが
できる。
なお、合成基質法エンドトキシン特異的リムルステスト試薬の製造法としては
、たとえば米国特許第４,４９５,２９４号明細書（Ｍethod for determining
bacterial endotoxin and kit therefor）で開示されている基質ならびにＲ
‐Ｉle‐Ｇlu‐Ａla‐Arg‐pＮＡ、methoxycarbonyl‐Ｄ‐hexahydrotyrosyl‐
Ｇly‐Ａrg‐pＮＡ・ＡcＯＨ等の基質を使用すれば、高感度の試薬が製造できる
。ここで、Ｒはアセチル基、α−Ｎ−ベンゾイル基、α−Ｎ−カルボベンゾキシ
基、Ｎ−tert−ブトキシカルボニル基、ｐ−トルエンスルホニル基、その他アミ
ノ酸Ｎ端保護基を表わす。
一例を示すと、ライセート０.０４mlにＭｇＣl2 １.５μgおよび合成
基質（Ｎ‐tert‐ブトキシカルボニル‐Ｌeu‐Ｇly‐Ａrg‐p‐ニトロアニリド
）４.０ μgを加えて凍結乾燥することによって製造することができる。この凍
結乾燥品に０.２Ｍ Ｔris‐ＨＣl緩衝液（pＨ８.０）０.１mlおよび検体０.１m
lを加えて３７℃、３０分間加温する時、エンドトキシンのみと反応し、黄色を
呈する。また、ゲル化法、比濁法、比濁時間分析法のためのＬＡＬ試薬は、ライ
セート０.１mlにＭgＣl2 １０.０μgを加えて凍結乾燥することによって製造す
ることができる。この試薬を参考例１−１、２−１、２−２と同様にして測定す
るとき、エンドトキシンのみと反応する。
産業上の利用可能性
以上述べたとおり、本発明のＧ因子活性化阻害剤は、ＬＡＬと組合わせること
により、エンドトキシン特異的ＬＡＬ−Ｔestを提供し、エンドトキシンの存在
の有無が明確でない感染症、敗血症を疑われている臨床検体を測定するときに特
に有用であり、真のグラム陰性菌感染症（エンドトキセミア）を的確に判別出来
る利点に加えるに、従来のＬＡＬ−Testを併用することにより真菌感染症を判別
出来ることから、感染菌タイプの早期判定による該感染菌への適切な治療薬剤の
選択、処置、並びにその治療効果の解析を可能とし、本発明の阻害剤を含むキッ
トの提供は診断、医療等医学の進歩に多大の寄与が期待できる。[Brief description of the drawings]
FIG. 1 is a molecular sieve (GPC) fractionation pattern of commercially available curdlan,
FIG. 2 shows a re-chromatographic fractionation pattern of Nos. 44 to 46 in FIG.
[Method for preparing factor G activation inhibitor of the present invention]
The factor G activation inhibitor of the present invention is prepared, for example, by the method shown in the following Preparation Examples.
Can be manufactured. In addition, among commercially available (1 → 3) -β-D-glucan, the scope of the present invention
Can be used as is.
Preparation Example 1:Preparation of commercial curdlan by molecular sieve chromatography
Curdlan (Wako Pure Chemical Industries, Reagent, Lot No. PEQ 9080, Mn> 136,
000, Mw / Mn> 2.76) 1 g of sample No. 101 was added to 0.3 M NaOH at 5 mg / ml.
100 μl each at room temperature under the following conditions.
Yon chromatography (GPC) was performed. ｛Column: TSKgelG6
000PWXLAnd G5000PWXL(Both 7.8 x 300 mm) are connected in series,
Mobile phase: 0.3 M NaOH, flow rate: 0.5 ml / min｝. The eluted low molecular fraction (
No. 44-46)
And chromatographed again to give a number average molecular weight of 3,050 and a polydispersity of 1.
29 samples (0.015 mg) were obtained (Sample No. 1). Attach the above GPC fractionation pattern
1 is shown in FIG. Further, a fractionation pattern obtained by rechromatizing fractions 44 to 46 in FIG.
FIG. 2 shows the components.
This sample No.1 was converted to β-1,3-glucanase (Zymolyase-100T,
And digested with GPC (column: TSKgelG4000P).
WXL, G3000PWXL, G2500PWXLIn-line; mobile phase: distilled water, flow rate: 0.
6ml / min) and analyzed the sugar composition (40% glucose,
30% Oose, 20% Laminaritriose, 8% Laminaritetraose, Lamina
(Lipentaose 2%, recovery rate 94%) was confirmed. From this, this sample (No.
The sugar structure of 1) is a β-polyg containing a (1 → 3) -β-D-glucoside structure.
It turns out that it is rucoside.
Preparation Example 2:Fractionation of curdlan based on solubility difference in water
50 g of a commercially available curdlan (sample No. 101) was suspended in distilled water, and the following flow system was used.
The fractionation was performed by the operation shown in the chart.
Preparation Example 3:Preparation of curdlan water-insoluble sugar fraction by formic acid degradation
45 g of sample No. 102 was obtained according to the method of K. Ogawa et al. [Carbohydr. Res.,29, 39
7-403 (1973)]. Follow the flow chart below
Shown inPreparation Example 4-1:Refractionation of the water-soluble fraction of curdlanic acid hydrolyzate by molecular sieve
0.15 g of the water-soluble fraction (sample No. 3) obtained in Preparation Example 3 shown above was distilled water 30
dissolved in GPC (column: TSK gelG3000PWXL× 2,
G2500PWXL× 1, each mobile phase: distilled water, flow rate 0.5 ml / min)
6 samples of different molecular weights (Nos. 11 to 16) collected by fractionation and re-chromatographed
I got
Preparation Example 4-2:Refractionation of water-insoluble fraction of curdlanic acid hydrolyzate by molecular sieve
0.2 g of the water-insoluble fraction obtained in Preparation Example 3 (sample No. 4) was added to 40 ml of 0.3 M Na
Dissolved in OH solution, and GPC (column: TSK gel G3000PWXL× 2, G2
500PWXL× 1, mobile phase: 0.3 M NaOH solution, flow rate 0.5 ml / min)
Fractionation and re-chromatography were performed in the same manner as in Preparation Example 4-1, and the eluate was 0.3 MH.
The mixture was neutralized by adding a Cl solution to obtain two kinds of samples having different molecular weights (Nos. 17 and 18).
Was.
Preparation Example 5:Preparation of samples from curdlan water-insoluble fraction by sonication
1 g of sample No. 102 was suspended in about 100 ml of 5 mM NaOH solution,
Wave generator, sonicatorTM(Otake Works, Model 5202PZT, Tokyo)
The molecular weight was reduced by sonication at 0 KHz and 80 w for 12 minutes.
The above preparation example was prepared by using 5 M NaOH as the treatment solution to make a final 0.3 M NaOH solution.
Chromatographic fractionation according to 4-2, and eight kinds of samples having different molecular weights (Nos. 19 to 22)
And 103 to 106).
Preparation Example 6-1:Preparation of inhibitory substances derived from seaweed (I)
Alame (Eisenia bicyclis) Is derived from T. Usui et al. Biol. Che
m.43, 603-611 (1979).
, Suita Shoten) After grinding 100g, extract low molecular soluble fraction with 80% ethanol
Removed and from the residue 2% CaClTwoUse aqueous solution
And extract the laminaran fraction. Next, the final concentration of the extract was determined using 95% ethanol.
The resulting precipitate was collected by centrifugation, washed with ethanol, and crude laminar
Obtain a sample. The crude sample was redissolved in distilled water and an anion exchanger (DEAE-Toyo) was used.
Pearl) to remove acidic substances (alginic acid, etc.) and pigments
Sample No. 25 was obtained from the re-precipitation.
Preparation Example 6-2:Preparation of seaweed-derived inhibitors (II)
MacombLaminaria japonicaThe sample of origin was JJ Connell et al., J. Chem. Soc.
, 3494 (1950), commercially available dried seaweed alga bodies (Suita Shoten, Tokyo)
) After grinding 100 g, the mixture was left standing and extracted with a 0.09 M HCl solution for about 3 days.
Separately, the filtrate was allowed to stand for another day, and a small amount of the resulting precipitate was removed by centrifugation.
Was added to 3 volumes of ethanol to make an approximately 75% solution, and the resulting precipitate was collected by centrifugation.
After washing with alcohol and drying, a water-soluble laminaran fraction (sample No. 27) was obtained.
Preparation Example 7-1:Preparation of fungal inhibitor (I)
fungusSclerotinia libertianaKitahara et al., Kidai Noriho
8, 100-105 (1957)SclerotinialibertianaFungus
The residue obtained by sufficiently extracting the defatted dry powder (30 g) with water is extracted with a 7% NaOH solution.
And extract 10% CuSOFourThe solution was added and precipitated, which was separated and added with hydrochloric acid.
Wash with acidic methanol to remove copper, wash with 80% methanol to remove HCl
Purification by repeating washing and drying three times with methanol, ether and 6 g of a sample
No. 28 was obtained.
Preparation Example 7-2:Preparation of fungal inhibitor (II)
fungusSchizophyllum commune: The sample from Suehirotake is commercially available
(Kaken Pharmaceutical: Sonifiran, Pharmaceutical: Lot No. J61
040) to K. Tabata et al., Carbohydr. Res.,89 121-135 (1981
) According to the procedure of Preparation Example 5 above, followed by sonication in an aqueous solution for 10 hours.
Three kinds of samples having different molecular weights by molecular sieve fractionation under potash conditions (No 29, 30, 3
1) was obtained.
Preparation Example 7-3:Preparation of fungal inhibitor (III)
yeastSaccharomyces cerevisiae: Β-glucan sample from baker's yeast is commercially available
90 mg of bread yeast glucan (Sigma Lot No. 56F-4027) and 50 m of distilled water
After stirring at room temperature for 2 hours, the mixture was centrifuged, and about 50 ml of the supernatant was concentrated under reduced pressure.
The volume was adjusted to 1 ml, and the insoluble material was removed by centrifugation again. A 0.64 mg sample (No. 33) was obtained from the supernatant.
Was.
Preparation Example 8:Preparation of samples from barley .BETA.-glucan
Commercially available barley β-glucan (Sigma, Lot No. 56F-0652) 0.3M
A 5 mg / ml solution was prepared with NaOH, and under alkaline conditions according to the above Preparation Example 4-2,
A β-glucan sample having a narrow molecular weight distribution (No. 36) was obtained by molecular sieve fractionation.
The commercially available barley β-glucan was dissolved in hot water at a concentration of 5 mg / ml,
Centrifuge (3,500 rpm, 10 minutes). Distilled water was added to the mobile phase according to Preparation Example 4-1.
GPC fractionation was collected 50 times in 100 μl aliquots and refractionated and collected under the same conditions.
Thus, two kinds of samples having different molecular weights (Sample Nos. 37 and 38) were obtained.
Preparation Example 9: PartCarboxymethylated (1 → 3) -β-D-glucan (degree of substitution DS =
Preparation of 0.63)
Curdlan water insolubles obtained according to Preparation Example 2 E. Clarke and B. A. Stone: P
hytochemistry1175-188 (1962).
And carboxymethylated. In other words, 100 g of curdlan water-insoluble matter was passed through a nitrogen stream
At 0 ° C. in 1 l of a 5M NaOH solution, and this is stirred with 236 g of molybdenum.
A solution obtained by dissolving nochloroacetic acid in 200 ml of water was added dropwise.
Stirred at 5 ° C for 2 hours. The resulting gel is stirred vigorously in 2.5 volumes of ethanol and thinned.
Powdered and filtered. Wash thoroughly with 70% ethanol, then ethanol, ether
Washed with ether and dried. This was dissolved in 7 l of water, neutralized with 1 M acetic acid,
Activated carbon (40 g) was added, the mixture was stirred at room temperature for 1 hour, and filtered. The filtrate was concentrated under reduced pressure to 1 liter.
And precipitated by adding 3 volumes of ethanol, washed with ethanol and ether,
Drying under reduced pressure over concentrated sulfuric acid gave 113.85 g.
The resulting curdlan partially carboxymethylated (1 → 3) -β-D-glucan is
D. F. Durso's Uranyl Nitrate Method in Carbobydrate Chem. VIII, 127-1
29 (1980), the degree of etherification (degree of substitution: Degree of Sub
(stitution: DS) was 0.63. This is glucose residue 1 which forms a sugar chain.
It means that 0.63 out of 3 replaceable hydroxyl groups per unit were replaced
Things.
25 mg of the obtained partially carboxymethylated (1 → 3) -β-D-glucan
GPC (column: Toyopearl) dissolved in 5 ml of 0.1 M aqueous ammonium acetate solution
HW65F, 5 × 100 cm; mobile phase: 0.1 M aqueous ammonium acetate solution; flow rate
: 5.8 ml / min), and GPC using another column (column: TS
KgelG6000PWXL+ G5000PWXL+ G3000PWXLUsed in series;
Mobile phase: 0.1 M ammonium acetate aqueous solution; flow rate: 0.6 ml / min)
A sample No. 41 (Mn = 231,000) having a narrow molecular weight distribution was obtained.
In addition, 0.3 g of partially carboxymethylated (1 → 3) -β-D-glucan was steamed.
Dissolve in 30 ml of distilled water and sonicate (9 kHz, 180-130 W, 1 hour, sonication
Kubota Seisakusho, Insonator Model 201M used as greige)
After adding 0.5 ml of 1 M aqueous ammonium acetate solution to 4.5 ml of the mixture and mixing.
GPC fractionation and collection were performed in the same manner as described above to obtain sample No. 41.
And GPC re-fractionation, and two kinds of samples having different molecular weights (Nos. 39 and 40)
I got
Preparation Example 10:Partial carboxymethylation of substitution degree 1.2 (1 → 3) -β-D-glu
Preparation of can
Carboxymethylation of the degree of substitution (DS) 0.63 obtained according to Preparation 9 (1
→ 3) 10 g of -β-D-glucan was added to 25 ml of 10.5M Na at 0 ° C under a nitrogen stream.
OH to make a paste, and with good stirring, add monochloroacetic acid aqueous solution (
10 g / 12 ml), warm to 60 ° C., stir for 4 hours, cool and then add 2M
HCl (30 ml) and then 200 ml of hydrochloric acid ethanol (40 ml HCl /
The resulting precipitate was collected by pouring into ethanol, washed with 70% ethanol, and then washed with ethanol.
The mixture was washed with an ether and ether, and dried under reduced pressure to obtain a sample of Sample No. 107.
Partial carboxymethylation of DS = 0.63 shown in Preparation Example 9 (1 → 3) -β-
The substitution degree was measured by the same method as that for D-glucan.
Atsuta.
Preparation Example 11:Preparation of partially carboxymethylated laminaran
Partially carboxymethylated laminaranLaminaria digitataLaminaran (Sig
Co., Ltd. Lot No. 77F-3885) and the partial carboxymethion of Preparation Example 9
As in the case of the E. Clarke and B. A. Stone: Phytoc
hem.1, 175 (1962), sample No. 42 (DS
= 0.06).
Preparation Example 12:Preparation of partially methylated (1 → 3) -β-D-glucan
3.0 g of a curdlan water-insoluble material obtained according to Preparation Example 2 was used. Samec, Kolloid-Beihe
fte51, 369 (1940), suspended in 80 ml of water and saturated under a stream of nitrogen.
1.35 ml of an aqueous solution of sodium hydroxide is added and completely dissolved.
60 g of sulfuric acid is gradually added, and after about 1 hour, the reaction solution is dropped into acetone to form
The precipitate was collected, thoroughly washed with acetone, dried over concentrated sulfuric acid under reduced pressure, and the title product (sample N
o.43, DS = 0.16).
Preparation Example 13:Preparation of partially sulfated laminaran Laminaria digitataSulfation of laminaran from pyridine
-3 sulfur oxide complex (Wako Pure Chemical Industries, Lot No. PPL8823)
I went as follows.
Well driedLaminaria digitataLaminaran (Sigma, Lot N)
o. 77F-3885) 0.5 g was dissolved in 50 ml of dehydrated pyridine and pyridine-
1 g of sulfur trioxide complex was added, reacted at 60 ° C. for 1 hour, and 100 ml of water was added.
And neutralized with NaOH, washed thoroughly with an aqueous alkaline solution to remove glucan.
Dialysis against water using the removed dialysis membrane (Spectropore 1,000 cuts)
Then, concentrate and add 2 volumes of acetone to precipitate the sugar component, and wash with acetone
Then, it was dried under reduced pressure over concentrated sulfuric acid to obtain 0.38 g of the title preparation (Sample No. 44, DS
= 0.14).
In addition, substitution of the methyl group and the sulfate group in each of the preparations shown in Preparation Examples 12 to 13
The degree was measured and calculated according to the method described in the following literature.
Ochiai, Tsuda, Sakamoto; Organic Quantitative Analysis (Micro Trace), Nanzando (1956);
Whistler, R .; L. ed., Methods in Carbohydrate Chemistry III, p229
-235,277-280 (1964), Academic Press
[Commercial sample]
After confirming the physical properties of the following commercially available samples, neutralize them as they are or after alkali solubilization
Provided.
Glucose: (Wako Pure Chemical Industries, JIS special grade reagent): Sample No. 108
Laminari-oligosaccharide: (Seikagaku Corporation, Pierre reagent): Sample Nos. 5 to 10
LaminaranEisenia araboreaOrigin: (Hanai Chemical, reagent): Sample No. 23
〃E.araboreaOrigin: (Tokyo Kasei, reagent): Sample No. 24
〃Laminaria digitataOrigin: (Sigma reagent): Sample No. 26
LentinanLentinus edodes(Shitake mushroom) Origin: (Yamanouchi Pharmaceutical, Pharmaceutical Lot
No.CKC7): Sample No.32
LihienanCetraria islandicaOrigin: (Sigma, reagent): Sample No.
34
〃Usnea barbataOrigin: (Sigma, reagent): Sample No. 35Examples 1-44
Table 2 below shows the measurement results of the molecular weight, factor G activation inhibition titer, and the like of each of the above samples.
You.
The molecular weight in the table is the number average molecular weight (Mn) defined by the following formula obtained by GPC.
), And the molecular weight distribution is represented by a polydispersity (Mw / Mn) defined by the following equation.
You.
Here, Hi is the i-th peak when the chromatogram is divided into equal parts by time.
The height of the sample (sample concentration) and Mi represent the i-th molecular weight.
Factor G activation inhibitory titers are shown below [Method for measuring activity titer of factor G activation inhibitor]
] And expressed as a unit per mg.
[Method for measuring activity titer of factor G activation inhibitor (hereinafter sometimes abbreviated as GI)]
The following are contained in 200 μl of the reaction mixture.
(1) Sample (Note 1) GI sample or pure water 50μl
[Factor G activator (abbreviated as GA, Note 2)] 10 pg added or not added
(2) horseshoe crab lysate clotting enzyme
Precursor fraction (A280= 2.5) (Note 3) 30μl
(3) horseshoe crab lysate factor G fraction
(A280= 0.9) (Note 3) 20 μl
(4) Tris-HCl buffer (pH 8.0) 20 μmole
(5) MgClTwo 20μmole
(6) Boc-Leu-Gly-Arg-pNA (t-butoxy
Carbonyl-L-leucyl-glycyl-L
-Arginine-p-nitroanilide) 0.13 μmole
After incubating the reaction solution at 37 ° C for 30 minutes, the released pNA (para
Nitroaniline) in 0.04% sodium nitrite (0.48 M HCl solution)
0.3% ammonium sulfamate, 0.07% N-1-naphthylethylenedi
Amine dihydrochloride (0.5 ml each) is added sequentially, and the color is obtained by diazo coupling.
Converted and absorbance at 545 nm (A545) Measure as quantity.
GI activity is calculated by the following equation.
Under these conditions, the amount of GI that inhibits the activation of factor G by 100% by GA is 1%.
00 units.
(Note 1) Of the samples, those insoluble in water are dissolved in 0.3 M NaOH and then made up to the same volume.
And neutralized by adding 0.3 M HCl.
(Note 2) GPC fractionated purified preparation of the sonicated curdlan prepared in Preparation Example 5 (
Table-2, No. 106, molecular weight 216,000).
(Note 3) Reference [T. Obayashi et al., Clin. Chim. Acta,149, 55-65 (
1985)] according to Japanese horseshoe crabT.tridentatusPrepared from
The following is clear from the contents of Table 2 above.
(A) In commercially available curdlan (sample No. 101) known as a factor G activator
Contains water-soluble low molecular weight factor G activation inhibitor
(Sample Nos. 1 and 2).
(B) Polyglycoside composed of (1 → 3) -β-D-glucoside structure
Of the (1 → 3) -β-D-glucoside structural units in the range of 2 to 370
The polyglycoside exhibits the factor G activation inhibitory effect of the present invention.
(C) High molecular weight β-glucan fraction with no inhibitory activity (sample No. 102)
Fractions having a molecular weight of 60,000 or less obtained by various low molecular weight reduction procedures
Factor activation inhibition titers were expressed (Sample Nos. 3, 4, 11 to 22).
(D) a poly- (1 → 3) -β-D-glucoside structure having a degree of polymerization of 10 or less
Large (1- → 4) -β-D-glucoside structure is linked in a block shape.
Wheat β-glucan [Ballance et al., Carbohyd. Res.,61, 107-118 (197).
8)] (Preparation Example 8, Sample Nos. 36, 37 and 38) are all laminaritetra.
Shows activity equivalent to ose or laminaripentaose, and the molecular weight of the whole glucan
Even if it is 60,000 or less, it can be used as the inhibitor of the present invention.
(E) Partial carboxymethyl of (1 → 3) -β-D-glucan obtained in Preparation Example 10
Samples having an average degree of substitution of 1.0 or more (Sample No. 107 DS = 1.
In the case of sample No. 2), the effect of inhibiting the activation of factor G disappeared. Raised as 26
Carboxymethylation (Preparative Example 11)
) To shorten the chain length of the (1 → 3) -β-D-glucoside moiety (Sample No.
. 42) and the sulfate modification of Preparation Example 13 to give (1 → 3) -β-D-glucose
It is also confirmed that the shortening of the side structure reduces the effect of inhibiting factor G activation.
Recognized
Was.
(F) the degree of polymerization of the (1 → 3) -β-D-glucoside structure portion is 370 or more;
(1 → 3) -β-glucan showing factor G activating effect with a molecular weight of 60,000 or more
, The partial methylation (Preparation Example 12, Sample No. 43),
The structure defined in the present invention by the chilling (Preparation Example 9, Sample No. 41)
In this case, it was confirmed that a factor G activation inhibitory effect was produced.
[Mode of action of the inhibitor of the present invention on factor G]
Among the horseshoe crab blood coagulation systems, the factor G activation system is described in T. Morita et al., F.
EBS Letters,129, 318-321 (1981).
As shown in the chart below. To clarify which part of the coagulation system the inhibitor of the present invention inhibits
The following experiment was performed.
A 200 μl reaction mixture contains a factor G activation inhibitor (laminariheptaose).
, Sample number 10, abbreviated as I) 5 μg, factor G activator (curdlan sonicated product)
GPC fraction, sample number 106, abbreviated as A) 3 pg, literature [T. Obayashi et al.
., Clin. Chim. Acta,149, 55-65 (1985)]
The prepared factor G fraction (abbreviated as G) 20
μl, 30 μl of coagulase precursor fraction (abbreviated as P), Tris-HCl buffer (pH 8.
0) 20 μmole, MgClTwo 20 μmole, synthetic substrate Boc-Leu-Gly-Arg-p
NA (abbreviated as S) including 0.13 μmole.
By changing the order of addition of each component and the heating conditions, the inhibition of the activation of the factor G
In each of the experiments (1 to 5), the degree when no inhibitor was added was used as a control.
The measurement results are shown below. As is clear from experiments 1, 2 and 3, factor G (precursor) was added to the inhibitor of the present invention (
When I) is added, regardless of the presence or absence of the factor G activator (A),
Offspring activation is 100% inhibited.
On the other hand, factor G was once activated by (A) as apparent from experiments 4 and 5.
After that, even if the inhibitor (I) is present, the inhibition of the active form factor G occurs.
No.
Therefore, the inhibitor (I) of the present invention is a substance acting only on factor G (precursor).
I can.
The amount of the inhibitor of the present invention that inhibits the activation of factor G present in LAL by 100%
If the substance (I) is present, no matter how much the amount of the activator (A) is present, the G factor
No activation of offspring occurs. However, small amounts of inhibitors that cannot inhibit factor G by 100%
When a large amount of the activator (A) is present in the presence of (I), the inhibitor (I)
It is also confirmed that factor G, which was not inhibited by the activator, is activated by the activator (A).
It was recognized.
This led to A. Kakinuma et al., Biochem. Biophys. Res. Commun.,1
01, 434-439 (1981), (1 → 3) -β-D-glucan
Derivatives and T. Morita et al. In Prog. Chim. Biol. Res.,189, 53-64 (
1985) on the ability to activate horseshoe crab factor G by various β-glucans.
The presence of the maximum activation concentration could be analyzed.
Example 45:Endotoxin-specific kit with or without factor G activation inhibitor
Measurement ability comparison
When the factor G activation inhibitor of the present invention is added to LAL-Test,
The endotoxin specificity comparison was performed using various samples by the following operation.
Was.
The LAL-Test product used was a Toxicolor test consisting of the following componentsTM(ratio
Color method, manufactured by Seikagaku Corporation).
Perchloric acid, sodium hydroxide, buffer, lysate + chromogenic synthetic substrate,
Endotoxin-free distilled water, 6 endotoxin standard products,
(1-Naphthyl) ethylenediamine dihydrochloride.
In the kit configuration, sample No. 13 was used as a factor G activation inhibitor in the buffer solution.
Using a solution of LAL at a concentration of 5 μg / ml to dissolve the LAL main reaction agent
Solution group (A-kit) and the solution dissolved using a buffer solution not containing the inhibitor.
Compare the reactivity of both A and B kits to various samples as a solution group (B-kit)
And shown in Table 3 below.
Note 1:Escherichia coli 0111: B4 derived endotoxin (Difco)
Note 2: Curdlan water-insoluble fraction; molecular weight 159,000 or more; Table 2, No. 102
* 3: Elastomers made of Kyupra ammonium rayon membrane, a regenerated cellulose membrane
Loaf fiber hemodialyzer AM-Neo-3,000 (made by Asahi Medical Co., Ltd.)
Washing solution obtained by perfusing with distilled water. The sugar content was measured by the phenol sulfuric acid method.
Note 4: Average ± standard deviation of 25 healthy subjects
Specimens b to e: suspected septic complication, blood cultureEscherichia coliIs detected
Was
f, g: Same as above,Pseudomonas aeruginosaWas detected
i: Same as above,Candida albicansWas detected
j: Same as above,Candida guilliermondiiWas detected
h: Pulmonary aspergillosis
k, l: cases diagnosed as systemic fungal infection at autopsy
m to r: Manufactured from Kyupura ammonium rayon membrane which is a regenerated cellulose membrane
Patients with chronic renal insufficiency (dialysis using microorganisms)
No infection due to
Specimens Nos. 1 to 5 were prepared by dissolving the specimens in a solvent according to the measurement manual of the Toxicolor Test.
The solution was dissolved as it was, and the reaction was carried out using 0.1 ml of the sample as a sample.
) Is the method of T. Obayashi [J. Lab. Clin. Med.,104, 321-330 (
1984)], after pre-treating the plasma sample using the above kit configuration.
Was used as a sample for the reaction.
The reactivity of each of the kits A and B was determined by using each sample prepared using the kit configuration.
The reaction was performed at 37 ° C for 30 minutes. The maximum reactivity in the case of this kit composition was ΔA545= 1.5.
As can be seen from Table-3, if the A-kit contains the factor G activation inhibitor of the present invention,
In addition, both the conventional product B-kit and the endotoxin sample (No. 1)
Although the results showed reactivity, the curdlan water-insoluble fraction, which is a factor G activator, was used.
(Sample No. 2) showed extremely high reactivity in the B-kit. on the other hand,
Although the sample showed no reactivity in the A-kit,
When combined with Syn (sample No. 3), the same reactivity as endotoxin of sample No. 1
showed that.
Cellulose known as Limulus test positive (non-endotoxin)
Dialysis membrane washing solution [FC Pearson et al., Artif. Organs,8, 291-2
98 (1984)] as specimens (No. 4, No. 5),
For both kit and B-kit, the above curdlan water-insoluble fraction and / or endotope
Similar results to the behavior in the case of adding toxin were shown.
Based on the above results, the factor G activation inhibitor of the present invention is used in combination with LAL.
This indicates that endotoxin-specific measurement is possible.
Furthermore, with the conventional LAL-Test, it is possible to clearly determine whether or not it is true endotoxemia.
Regarding the clinical blood sample that was excluded, the A-kit according to the present invention and the conventional B-key
As a result of the bacterial culture, the presence of Gram-negative bacteria was confirmed (No.
6; b to g), both A and B kits showed high reactivity, while (
Existence of fungi known to have 1 → 3) -β-D-glucan on the cell wall of cells
The reactivity of A-kit was not shown in the specimens whose presence was confirmed (same as: hl)
, B-kit showed high reactivity. In addition, from clinical symptoms,
A in hemodialysis patients (chronic renal failure) that are not considered
-No reactivity was observed with the kit, and the abnormally high value with the B-kit was due to dialysis membrane-derived (1 →
3) It was predicted to be due to -β-D-glucan.
By combining LAL-Test and the factor G activation inhibitor of the present invention,
Suspicion of infectious disease or sepsis whose presence or absence of endotoxin is unclear
Measuring clinical specimens can be a true gram-negative bacterial infection (endotoxemia)
In addition to the advantage of being able to reliably identify fungal infections, the ability to identify fungal infections
Selection and treatment of an appropriate therapeutic agent for the infectious bacteria by early determination of
It is possible to analyze the effect and provide a kit containing the inhibitor of the present invention.
Can greatly contribute to the progress of
Hereinafter, as a reference example, the factor G activation inhibitor of the present invention and horseshoe crab Amebosa
Of a kit for endotoxin-specific detection and measurement using a combination of kit and lysate
Here is an example.
Reference Example 1:Commercially available factor G activation inhibitor or existing Limulus test reagent To the endotoxin-specific measurement.
How to make a kit
1-1. Limulus test reagent (lyophilized product) can be used as specified
(Distilled water or buffer), and add the factor G activation inhibitor of the present invention to the same as the sample.
Those who achieve the purpose by adding at the same time or separately (in any order)
Law:
For example, "Pregel S" (freeze-dried product; gel method Limulus test product;
0.1 ml of distilled water was added to and dissolved in 0.1 ml of distilled water.
GPC fraction Fraction 4: 0.01 ml (1 μg / ml) of an aqueous solution (1.2 μg / ml) of Table 2, No. 14)
20 μg / ml LAL) and 0.1 ml of the sample were added and gently shaken.
Reacts with only endotoxin when left standing and warming for 1 minute to form a gel.
1-2. Dissolve the inhibitor in advance in a Limulus test reagent solution and use it
How to achieve your goal by dissolving Limulus Test Reagent:
For example, Cotest Endotoxin (a synthetic substrate method Limulus Test product:
If you use Kirby Bitram, 1 vial of LAL (lyophilized product)
A steam in which 0.7 μg of the inhibitor (laminaran; No. 26 in Table 2) was previously dissolved.
Dissolve in 1.4 ml of distilled water. 0.1 ml of the sample is added to 0.1 ml (500 ng / ml LAL).
In addition, the mixture was heated at 37 ° C. for 10 minutes, and 0.2 ml of a buffer containing a synthetic substrate (S-2423) was added.
When added and heated at 37 ° C for 3 minutes, it reacts only with endotoxin and shows yellow
You. For the determination, 200 μl of a 50% acetic acid solution was added, and the absorbance was measured at 405 nm.
Measure.
Reference Example 2:By pre-adding factor G activation inhibitor to LAL, How to make an endotoxin-specific Limulus test reagent kit
2-1. Preliminary inhibition with commercially available LAL (so-called gelling Limulus test reagent)
How to achieve the purpose by adding harmful agents:
For example, "Limulus HS-Test Wako" (freeze-dried product; gel method
When the product is measured by turbidimetric analysis using Wako Pure Chemical Industries, Ltd.
, One vial of LAL (lyophilized product) was added to the inhibitor (curdrangic acid) in advance.
GPC fraction of degraded product Fraction 4; in Table 2, No. 14) 5 ml of distilled water in which 0.5 μg was dissolved
Dissolve with. Dispense 0.1 ml (100 ng / ml LAL) into a test tube for reaction,
Further, add 0.1 ml of the sample, shake gently, and use a turbidimetric time analyzer “Toxinome
ET-201 ”(Wako Pure Chemical Industries) dedicated analysis module (37 ° C)
At the predetermined photometry position, and turn on the start switch. Endotoxin only
Reacts and the gel time is displayed.
2-2. Adding the inhibitor to a previously prepared LAL before adding the sample
How to reach the goal by:
For example, when measuring by turbidimetry using LAL, horseshoe crab (Tachypl
eus tridentatus) 0.1m lysate extracted from blood cells using hypotonic buffer
1M in which 0.5 μg of the inhibitor (laminaran; No. 26 in Table 2) was dissolved per ml
Tris-HCl-1M MgClTwo0.01 ml of a buffer (pH 8.0) solution (50 ng / m2)
(LAL). To this is added 0.1 ml of the sample, and the mixture is heated at 37 ° C. Endo
Only the toxin reacts and becomes cloudy. If you measure the absorbance at 660nm over time,
Wear.
Reference Example 3:By adding a factor G activation inhibitor during the preparation of LAL extraction,
Manufactures lysate as raw material for londolus test reagent how to
For example, horseshoe crab (Tachypleus tridentatus,T. gigas,Limulus pol
yphemus,Carcinoscorpius rotundicauda Blood lymph)
The cells were collected and centrifuged to obtain blood cells (about 20 g), to which 2.0 mg / l of the inhibitor (parts) was added.
Carboxymethylated laminaran; in Table 2, No. 42) in distilled water or 0.02
Dissolution of the inhibitor in a hypotonic buffer such as M Tris-HCl buffer (pH 8.0)
Add 100 ml of the solution, homogenize with a Waring blender, and centrifuge
(8,000 rpm; 30 minutes; 4 ° C.) to fractionate into supernatant and precipitate. This operation
Repeat once to obtain a total of about 150 ml of supernatant as LAL. This LAL constant
Limulus test reagent (gelation method)
, Turbidimetric method, turbidimetric time analysis method, synthetic substrate method)
To produce endotoxin-specific Limulus test reagents that react specifically
it can.
The synthetic substrate method for the production of endotoxin-specific Limulus test reagents includes:
For example, U.S. Pat. No. 4,495,294 (Method for determining).
substrates and R disclosed in bacterial endotoxin and kit therefor)
-Ile-Glu-Ala-Arg-pNA, methoxycarbonyl-D-hexahydrotyrosyl-
Use of a substrate such as Gly-Arg-pNA / AcOH can produce a highly sensitive reagent
. Here, R is an acetyl group, α-N-benzoyl group, α-N-carbobenzoxy
Group, N-tert-butoxycarbonyl group, p-toluenesulfonyl group,
Represents an N-terminal protecting group for the acid.
As an example, 0.04 ml of lysate was added to MgClTwo 1.5 μg and synthetic
Substrate (N-tert-butoxycarbonyl-Leu-Gly-Arg-p-nitroanilide
) It can be produced by adding 4.0 μg and freeze-drying. This freeze
0.1 ml of 0.2 M Tris-HCl buffer (pH 8.0) and 0.1 m of sample
When the mixture is heated at 37 ° C for 30 minutes, it reacts only with endotoxin and turns yellow.
Present. LAL reagents for gelation, turbidimetry and turbidimetric time analysis are
MgCl in 0.1 ml of sateTwo It is prepared by adding 10.0 μg and freeze-drying.
Can be This reagent is measured in the same manner as in Reference Examples 1-1, 2-1 and 2-2.
Reacts only with endotoxin.
Industrial applicability
As described above, the factor G activation inhibitor of the present invention may be used in combination with LAL.
Provides an endotoxin-specific LAL-Test and the presence of endotoxin
When measuring clinical specimens suspected of infectious disease or sepsis whose presence or absence is unclear.
And can accurately identify true gram-negative bacterial infections (endotoxemia)
In addition to the advantages described above, the fungal infection can be identified by using the conventional LAL-Test.
Because it is possible, an appropriate therapeutic drug for the infectious bacteria can be determined by early determination of the infectious bacteria type.
It allows selection, treatment, and analysis of its therapeutic effects, and includes kits containing the inhibitors of the present invention.
The provision of medical services can be expected to greatly contribute to the advancement of medicine such as diagnosis and medical treatment.
Claims (1)
合したポリ−（１→３）−β−Ｄ−グルコシド構造部分を少なくとも１つ含有す
る ａ） 分子量３４２〜１,６３８のラミナリオリゴ糖、 ｂ） 分子量１,８００〜３,２５８のラミナリデキストリン、および ｃ） 上記式（Ｉ）で示される（１→３）−β−Ｄ−グルコシド構造単位のみ
から実質的になる実質的に直鎖状で平均分子量２,０００〜６０,０００の（１→
３）−β−Ｄ−グルカン よりなる群から選ばれるポリグリコシドであって、Ｇ因子活性化物質、カブトガ
ニ・アメボサイト・ライセート凝固酵素前駆体画分、カブトガニ・アメボサイト
・ライセートＧ因子画分、緩衝剤、塩化マグネシウムおよび発色合成基質を阻害
剤検体に添加した後、一定時間加温する間に 遊離する発色基を定量することにより測定されるＧ因子活性化阻害力価が少なく
とも６,３１０,０００単位／ｍｇであるポリグリコシド を有効成分として含有す
ることを特徴とするカブトガニ・アメボサイト・ライセートＧ因子活性化阻害剤
。 ２．ポリグリコシドが、カードランの部分分解物または部分分解物の分画物で
ある請求の範囲第１項記載の阻害剤。 ３．ポリグリコシドが、カードランの部分分解物または部分分解物の分画物で
あり、そしてＧ因子活性化物質、カブトガニ・アメボサイト・ライセート凝固酵
素前駆体画分、カブトガニ・アメボサイト・ライセートＧ因子画分、緩衝剤、塩
化マグネシウムおよび発色合成基質を阻害剤検体に添加した後、一定時間加温す
る間に遊離する発色基を定量することにより測定されるＧ因子活性化阻害力価が
少なくとも６,３１０,０００単位／ｍｇのポリグリコシドである請求の範囲第１
項記載の阻害剤。 ４．請求の範囲第１項記載のポリグリコシドの有効量を、カブトガニ・アメボ
サイト・ライセートに添加することからなる該カブトガニ・アメボサイト・ライ
セート中に存在することがあるＧ因子の活性化を阻害する方法。 ５．請求の範囲第１項記載のポリグリコシドの有効量を含有するエンドトキシ
ンに特異的なリムルステスト試薬。 ６．請求の範囲第１項記載のポリグリコシドを、カブトガニ・アメボサイト・
ライセート１ｍｌ当り少なくとも５０ｎｇ含有するエンドトキシンに特異的なリ
ムルステスト試薬。[Claims] 1. formula A) Molecular weight containing at least one poly- (1 → 3) -β-D-glucoside structural part in which 2 to 370 (1 → 3) -β-D-glucoside structural units are continuously bonded. 342 to 1,638 laminarioligosaccharides, b) laminaridextrin having a molecular weight of 1,800 to 3,258, and c) only the (1 → 3) -β-D-glucoside structural unit represented by the above formula (I).
(1 →) having a substantially linear average molecular weight of 2,000 to 60,000
3) A polyglycoside selected from the group consisting of -β-D-glucan, which comprises a factor G activator,
D. amebosite lysate coagulase precursor fraction, horseshoe crab amebosite
・ Inhibits lysate factor G fraction, buffer, magnesium chloride and chromogenic synthetic substrate
The factor G activation inhibitory titer, which is measured by quantifying the chromophoric group released during heating for a certain period of time after addition to the agent sample, is low.
A horseshoe crab, amebosite, lysate factor G activation inhibitor, comprising as an active ingredient a polyglycoside that is at least 6,310,000 units / mg . 2 . 2. The inhibitor according to claim 1, wherein the polyglycoside is a partially degraded product of curdlan or a fraction of a partially degraded product. 3 . The polyglycoside is a partially degraded product or a fraction of a partially degraded product of curdlan, and a factor G activator, a horseshoe crab amebosite lysate coagulase precursor fraction, a horseshoe crab amebosite lysate factor G fraction, After adding a buffer, magnesium chloride and a chromogenic synthetic substrate to the inhibitor sample, the factor G activation inhibitory titer, determined by quantifying the chromophoric group released during heating for a certain period of time, is at least 6,310, 000 units / mg of polyglycoside.
The inhibitor according to Item. 4 . A method for inhibiting the activation of factor G which may be present in a horseshoe crab amebosite lysate, comprising adding an effective amount of the polyglycoside according to claim 1 to a horseshoe crab amebosite lysate. 5 . A limulus test reagent specific for endotoxin, comprising an effective amount of the polyglycoside according to claim 1. 6 . The polyglycoside according to claim 1 is used for horseshoe crab, amebosite,
Limulus test reagent specific for endotoxin containing at least 50 ng per ml of lysate.
Family
ID=
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