JP4484151B2 - Human blood group reagent - Google Patents
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- JP4484151B2 JP4484151B2 JP2005092784A JP2005092784A JP4484151B2 JP 4484151 B2 JP4484151 B2 JP 4484151B2 JP 2005092784 A JP2005092784 A JP 2005092784A JP 2005092784 A JP2005092784 A JP 2005092784A JP 4484151 B2 JP4484151 B2 JP 4484151B2
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Description
本発明は、ヒト血液型試薬に関し、詳しくは、キノコ、特にブナシメジから精製されるマイコグリコリピド（ＡＧＬと略す。キノコの酸性糖脂質あるいはイノシトールリン酸性糖脂質とも称される。）を類似ヒト血液型抗原として使用するヒト血液型試薬に関するものである。 The present invention relates to a human blood group reagent, and more specifically, mushrooms, particularly mycoglycolipid purified from beech shimeji (abbreviated as AGL. Mushroom acidic glycolipid or inositol phosphoric acid glycolipid) is also similar to human blood. The present invention relates to a human blood group reagent used as a type antigen.
近年の急速な高齢化社会の進展と食生活の変化による生活習慣病の増加は、健康への関心を高め、キノコの生理活性が注目されている。
キノコは生活習慣病を始めとして癌や慢性関節リュウマチ、アトピー性皮膚炎などの疾病に効果があるとされ、食用キノコに含まれる新たな有用物質の探索やその生理活性に興味を持たれている。
The recent progress of an aging society and an increase in lifestyle-related diseases due to changes in dietary habits have raised interest in health, and the physiological activity of mushrooms has attracted attention.
Mushrooms are said to be effective in diseases such as lifestyle-related diseases, cancer, rheumatoid arthritis, and atopic dermatitis, and are interested in exploring new useful substances contained in edible mushrooms and their physiological activities. .
特にキノコのＡＧＬの生理活性としては、イノシトールリン酸セラミド型の糖脂質がマウスへの免疫実験において、毒性のない良好なアジュバンド活性を有することやヒトや動物の血清中に一部のイノシトールリン酸セラミド型糖脂質を認識する抗体が存在することなどが報告されている。 In particular, the physiological activity of mushroom AGL is that inositol phosphate ceramide-type glycolipids have good adjuvant activity without toxicity in immunization experiments on mice, and some inositol phosphorus in human and animal sera. It has been reported that an antibody that recognizes acid ceramide type glycolipid exists.
キノコの中でもブナシメジには、特にＡＧＬが多く含まれ、このブナシメジから精製されるＡＧＬの有効活用が望まれる。 Among mushrooms, beech shimeji is particularly rich in AGL, and effective utilization of AGL purified from this beech shimeji is desired.
本発明者は、このブナシメジから精製されるＡＧＬにつき、更に鋭意検討研究した結果、このＡＧＬの有効活用方法を明らかにした。
本発明の目的は、ブナシメジから精製されるＡＧＬを生理活性物質として有効活用することにある。 An object of the present invention is to effectively utilize AGL purified from Bunashimeji as a physiologically active substance.
本発明者は、ブナシメジに含まれるマイコグリコリピドに含有される化合物について、鋭意検討を重ねた結果、ヒト血液型Ｏ型抗原構造、Ｂ型抗原構造、Ａ型抗原構造を有することを見出し、本発明に至った。 As a result of intensive studies on the compound contained in mycoglycolipid contained in Bunashimeji, the present inventor has found that it has a human blood group O type antigen structure, a B type antigen structure, and an A type antigen structure. Invented.
第1の本発明に係る、ブナシメジから精製される構造式：Ｇａ１α１−３（Ｆｕｃα１−２）Ｇａ１β１−６Ｍａｎα１−２Ｉｎｓ−Ｐ−Ｃｅｒから成るＢ型糖脂質のマイコグリコリピドは、Ｇａ１α１−３（Ｆｕｃα１−２）Ｇａ１の構造式を有しており、この構造式はＢ型抗原構造で、ヒト血液型判定の試薬として安価に利用することができ、ブナシメジのマイコグリコリピドの有効活用ができる。 The mycoglycolipid of type B glycolipid consisting of Ga1α1-3 (Fucα1-2) Ga1β1-6Manα1-2Ins-P-Cer, which is purified from Bunashimeji, according to the first invention is Ga1α1-3 (Fucα1 -2) It has a structural formula of Ga1, and this structural formula is a B-type antigen structure, which can be used at low cost as a reagent for human blood group determination, and can effectively use the mycoglycolipid of Bunashimeji.
第2の本発明に係る、ブナシメジから精製される構造式：Ｆｕｃα１−２Ｇａ１β１−６Ｍａｎα１−２Ｉｎｓ−Ｐ−Ｃｅｒから成るＯ型糖脂質のマイコグリコリピドは、Ｆｕｃα１−２Ｇａ１の構造式を有しており、この構造式はＯ型抗原構造で、ヒト血液型判定の試薬として安価に利用することができ、ブナシメジのマイコグリコリピドの有効活用ができる。 The mycoglycolipid of the O-type glycolipid consisting of Fucα1-2Ga1β1-6Manα1-2Ins-P-Cer, which is purified from Bunashimeji, according to the second present invention has the structural formula of Fucα1-2Ga1 This structural formula is an O-type antigen structure, which can be used at low cost as a reagent for determining human blood group, and can effectively use mycoglycolipid of Bunashimeji.
第3の本発明に係る、ブナシメジから精製される構造式：Ｆｕｃα１−２Ｇａ１β１−６Ｍａｎα１−２Ｉｎｓ−Ｐ−Ｃｅｒから成るＯ型糖脂質のマイコグリコリピドに、人胃癌由来の細胞株のＡ型糖転移酵素によりＡ型糖脂質のマイコグリコリピドを合成することができるので、ヒト血液型判定の試薬として安価に利用することができ、ブナシメジのマイコグリコリピドの一層の有効活用ができる。 According to the third aspect of the present invention, the mycoglycolipid of O-type glycolipid consisting of structural formula: Fucα1-2Ga1β1-6Manα1-2Ins-P-Cer purified from Bunashimeji is transferred to A-type glycosyltransferase of a cell line derived from human stomach cancer. Since the mycoglycolipid of type A glycolipid can be synthesized by an enzyme, it can be used at low cost as a reagent for human blood group determination, and the mycoglycolipid of Bunashimeji can be used more effectively.
本発明に係る第1、2の発明であるマイコグリコリピドを見出した分析方法につき説明する。
これまでに報告されているキノコの酸性糖脂質（マイコグリコリピドあるいはイノシトールリン酸性糖脂質とも称される）の化学構造は、マンノース−イノシトールリン酸セラミド（Ｍａｎ−Ｉｎｓ−Ｐ−Ｃｅｒ）をコアーとして、更に、ガラクトースやフコース等が結合したものである。
An analysis method for finding the mycoglycolipids according to the first and second inventions of the present invention will be described.
The chemical structure of mushroom acidic glycolipid (also called mycoglycolipid or inositol phosphoric acid glycolipid) reported so far is based on mannose-inositol phosphate ceramide (Man-Ins-P-Cer) as the core. Furthermore, galactose, fucose and the like are combined.
以下に構造解析を行ったブナシメジのマイコグリコリピドについて述べる。
市販のブナシメジを５０℃で乾燥後、クロロホルム−メタノール−水（６０：３５：８）により総脂質を抽出した。
The following is a description of the mycoglycolipid of Bunashimeji which has been subjected to structural analysis.
After drying commercially available bunashimeji at 50 ° C., total lipid was extracted with chloroform-methanol-water (60: 35: 8).
総脂質を弱アルカリ及び弱酸性処理して調整した粗スフィンゴ脂質画分をイオン交換セファデックスカラムクロマトグラフィーによって分画した。
得られた酸性糖脂質画分をイアトロビーズカラムクロマトグラフィーに処し、7種のマイコグリコリピド（ＴＬＣ上での移動度の大きい順にＡＧＬ−０〜ＡＧＬ−６と仮称）をそれぞれ単離、精製した。
The crude sphingolipid fraction prepared by treating the total lipid with a weak alkali and a weak acid was fractionated by ion-exchange Sephadex column chromatography.
The obtained acidic glycolipid fraction was subjected to iatrobead column chromatography to isolate and purify 7 kinds of mycoglycolipids (tentative names AGL-0 to AGL-6 in descending order of mobility on TLC). did.
カラムからの溶出は、クロロホルム−メタノール−３Ｍアンモニア（７０：３０：３〜５０：５０：１７）の濃度勾配溶出法及びｎ−プロパノール−水−アンモニア（７５：１５：５及び７５：２０：５）の単一溶媒溶出法を用いた。 Elution from the column was performed using gradient elution method of chloroform-methanol-3M ammonia (70: 30: 3-50: 50: 17) and n-propanol-water-ammonia (75: 15: 5 and 75: 20: 5). ) Single solvent elution method.
単離した７種のマイコグリコリピドは、いずれもリン検出試薬のＨａｎｅｓ−Ｉｓｈｅｒｗｏｏｄ試薬に陽性を示し、更に、ＡＧＬ−１〜ＡＧＬ−６はｏｒｃｉｎｏｌ−硫酸試薬にも陽性であった（図１、図２）。 The seven isolated mycoglycolipids were all positive for the phosphorus detection reagent Hanes-Isherwood reagent, and AGL-1 to AGL-6 were also positive for the orcinol-sulfuric acid reagent (FIG. 1, Figure 2).
構成糖種はメチルグリコシドＴＭＳ−誘導体として、糖鎖結合位置は部分メチル化アルジトールアセテート誘導体として、それらのＧＣ及びＧＣ−ＭＳ分析によって決定した（図3）。 The constituent sugar species were determined as methylglycoside TMS-derivatives and the sugar chain binding positions as partially methylated alditol acetate derivatives as determined by GC and GC-MS analysis (FIG. 3).
更に、イノシトールリン酸部分の構造を酸加水分解生成物からのイノシトールリン酸及びイノシトールの同定と、フッ化水素酸による分解成績体からのセラミドの回収及びその解析によって確認した。 Furthermore, the structure of the inositol phosphate part was confirmed by the identification of inositol phosphate and inositol from the acid hydrolysis product, and the recovery and analysis of ceramide from the decomposition product by hydrofluoric acid.
また、マンノースが結合するイノシトールの水酸基の位置（Ｍａｎ→Ｉｎｓ）は、主として過ヨウ素酸酸化によるフラグメント成績体の構造解析の結果により決定した。 Further, the position of the hydroxyl group of inositol to which mannose binds (Man → Ins) was determined mainly by the result of structural analysis of the fragment product by periodate oxidation.
一方、ＭＡＬＤＩ−ＴＯＦ ＭＳ分析では、セラミド組成を考慮した推定分子量に相当する分子量ピークが得られた。
即ち、7種のマイコグリコリピドは、ＡＧＬ−０に順次マンノース（ｍ／ｚ162）、ガラクトース（ｍ／ｚ162）、フコース（ｍ／ｚ148）、ガラクトース（ｍ／ｚ162）、ガラクトース（ｍ／ｚ162）及びガラクトース（ｍ／ｚ162）分に相当する分子量の増加することがわかった（図4）。
On the other hand, in the MALDI-TOF MS analysis, a molecular weight peak corresponding to the estimated molecular weight considering the ceramide composition was obtained.
That is, seven kinds of mycoglycolipids are AGL-0, mannose (m / z 162), galactose (m / z 162), fucose (m / z 148), galactose (m / z 162), galactose (m / z 162) and It was found that the molecular weight corresponding to galactose (m / z 162) increased (FIG. 4).
糖のアノマー配置は１Ｈ−ＮＭＲ分析によったが、１つのガラクトースがβ−アノマーである他は、すべてα−アノマーであった（図5）。
以上のことから、ブナシメジの７種のマイコグリコリピドの構造を次のように決定した。
The anomeric configuration of the sugars was determined by 1 H-NMR analysis, but all were α-anomers except that one galactose was a β-anomer (FIG. 5).
From the above, the structure of seven mycoglycolipids of Bunashimeji was determined as follows.
ＡＧＬ−０：Ｉｎｓ−Ｐ−Ｃｅｒ； ＡＧＬ−１：Ｍａｎα１−２Ｉｎｓ−Ｐ−Ｃｅｒ； ＡＧＬ−２：Ｇａ１β１−６Ｍａｎα１−２Ｉｎｓ−Ｐ−Ｃｅｒ； ＡＧＬ−３：Ｆｕｃα１−２Ｇａ１β１−６Ｍａｎα１−２Ｉｎｓ−Ｐ−Ｃｅｒ； ＡＧＬ−４：Ｇａ１α１−３（Ｆｕｃα１−２）Ｇａ１β１−６Ｍａｎα１−２Ｉｎｓ−Ｐ−Ｃｅｒ； ＡＧＬ−５：Ｇａ１１−２Ｇａ１１−３（Ｆｕｃ１−２）Ｇａ１１−６Ｍａｎ１−２Ｉｎｓ−Ｐ−Ｃｅｒ； ＡＧＬ−６：Ｇａ１１−２Ｇａ１１−２Ｇａ１１−３（Ｆｕｃ１−２）Ｇａ１１−６Ｍａｎ１−２Ｉｎｓ−Ｐ−Ｃｅｒ。 AGL-0: Ins-P-Cer; AGL-1: Manα1-2Ins-P-Cer; AGL-2: Ga1β1-6Manα1-2Ins-P-Cer; AGL-3: Fucα1-2Ga1β1-6Manα1-2Ins-P- AGL-4: Ga1α1-3 (Fucα1-2) Ga1β1-6Manα1-2Ins-P-Cer; AGL-5: Ga11-2Ga11-3 (Fuc1-2) Ga11-6Man1-2Ins-P-Cer; AGL- 6: Ga11-2Ga11-2Ga11-3 (Fuc1-2) Ga11-6Man1-2Ins-P-Cer.
セラミド組成については、これらのマイコグリコリピドのすべてが、長鎖塩基はフィトスフィンゴシンのみを成分とし、一方、脂肪酸はＡＧＬ−０がＣ１６及びＣ１８の飽和酸とＣ１８，Ｃ２２，Ｃ２４の２−ヒドロキシ酸のそれぞれを成分とするのに対し、ＡＧＬ−１〜ＡＧＬ−６は、２−ヒドロキシ酸のみを構成成分としていた（図6）。 As for the ceramide composition, all of these mycoglycolipids are composed of only phytosphingosine as a long-chain base, while fatty acids are C16 and C18 saturated acids and C18, C22 and C24 2-hydroxy acids with AGL-0. Each of AGL-1 to AGL-6 has only 2-hydroxy acid as a component (FIG. 6).
これらのマイコグリコリピドの構造を、既に報告されているブナシメジと分類学上で同目のツクリタケのそれら（Ｇａ１α１−６Ｇａ１α１−６Ｇａ１α１−６（Ｆｕｃα１−２）Ｇａ１β１−６Ｍａｎα１−２Ｉｎｓ−Ｐ−Ｃｅｒ）と比較したところ、構成糖種は同一であるが、非還元端末からのガラクトースの結合に違いが認められた。 The structure of these mycoglycolipids is the same as that of Tsunatake, which has the same taxonomy as the previously reported bunashimeji (Ga1α1-6Ga1α1-6Ga1α1-6 (Fucα1-2) Ga1β1-6Manα1-2Ins-P-Cer) When compared, the constituent sugar species were the same, but a difference was observed in the binding of galactose from the non-reducing terminal.
ツクリタケを含めタマゴテングダケ、ホコリタケ、ヤマドリタケ、アンズタケ、シイタケ、ヒラタケ等、今までに報告されているマイコグリコリピドは、全てに共通した骨格構造であるイノシトールリン酸セラミドにマンノースがα１−２結合している。 Mycoglycolipids that have been reported so far, such as octopus agaric, mushroom, madoritake, chanterelles, shiitake, oyster mushrooms, etc., have mannose α1-2 linked to inositol phosphate ceramide, which is a common skeletal structure. Yes.
そして更に、マンノース、ガラクトース及び分岐でフコースが結合して、それらの組み合わせによって糖鎖が延長している。 In addition, mannose, galactose and branching fucose are combined, and the sugar chain is extended by the combination thereof.
ブナシメジを含め、前述のエリンギにおいても、それぞれのキノコを特色付けるマイコグリコリピドが存在することは、それらを食用として摂取する動物（特に、ヒト）に及ぼす影響等、生理活性についても関心が寄せられるところである。 The presence of mycoglycolipids that characterize each mushroom in the above-mentioned eringi, including buna shimeji, is also of interest for physiological activities such as the effects on animals (especially humans) that ingest them as food. By the way.
また、ブナシメジのマイコグリコリピドの糖鎖の非還元末端構造に注目すると、ＡＧＬ−３のＦｕｃα１−２Ｇａ１はヒト血液型のＯ型抗原構造を有し、ＡＧＬ−４のＧａ１α１−３（Ｆｕｃα１−２）Ｇａ１はＢ型抗原構造を有している。 Further, when attention is paid to the non-reducing terminal structure of the sugar chain of mycoglycolipid of Bunashimeji, FUCα1-2Ga1 of AGL-3 has a human blood type O-type antigen structure, and Ga1α1-3 (Fucα1-2 of AGL-4). ) Ga1 has a B-type antigen structure.
ＴＬＣ上でのそれぞれの抗体との反応性を調べるＴＬＣ−ｉｍｍｕｎｏｓｔａｉｎｉｎｇでは、ＡＧＬ−４は血液型判定抗血清及び抗体あるいはＡ型の血液型血清中に含まれる抗Ｂ抗体により認識された。
一方、ＡＧＬ−３は、Ｏ型糖鎖を認識するレクチンにより検出された。
In TLC-immunostaining, which examines the reactivity with each antibody on TLC, AGL-4 was recognized by blood group determination antiserum and antibody or anti-B antibody contained in type A blood group serum.
On the other hand, AGL-3 was detected by a lectin that recognizes an O-type sugar chain.
第3の本発明に係る一実施例を説明する。
最初の、材料としては、人胃癌由来の細胞株、Ｈ型糖脂質（ＡＧＬ−３）、Ｆｕｃα１−２Ｇａ１β１−６Ｍａｎα１−２Ｉｎｓ−Ｐ−Ｃｅｒ、Ｂ型糖脂質（ＡＧＬ−４）、Ｇａ１α１−３（Ｆｕｃα１−２）Ｇａ１β１−６Ｍａｎα１−２Ｉｎｓ−Ｐ−Ｃｅｒ、ＵＤＰ−ＧａｌＮＡｃ（糖転移酵素反応のドナー基質）、薄層プレート、抗Ａ型抗体（モノクローナル抗体、ダコ社製）、セパックＣ１８逆層クロマトカートリッジを用いる。
An embodiment according to the third aspect of the present invention will be described.
As the first material, cell lines derived from human stomach cancer, H-type glycolipid (AGL-3), Fucα1-2Ga1β1-6Manα1-2Ins-P-Cer, B-type glycolipid (AGL-4), Ga1α1-3 ( Fucα1-2) Ga1β1-6Manα1-2Ins-P-Cer, UDP-GalNAc (donor substrate for glycosyltransferase reaction), thin layer plate, anti-A type antibody (monoclonal antibody, manufactured by Dako), Sepak C18 reverse layer chromatography cartridge Is used.
次に、方法に付き説明する。
細胞培養
上記の２種の細胞株をＲＰＭＩ１６４０／１０％ＦＣＳで培養し、４Ｘ１０７細胞を得る。
この細胞をホモジネート処理し、これを酵素源とする。比活性を上げるために１００．０００Ｇ上清のミクロゾームリッチ画分を用いる。
Next, the method will be described.
Cell Culture The above two cell lines are cultured in RPMI 1640/10% FCS to obtain 4 × 10 7 cells.
The cells are homogenized and used as an enzyme source. Use a microsomal rich fraction of 100.000 G supernatant to increase specific activity.
酵素反応
基質：Ｈ型糖脂質（ＡＧＬ−３） １０ｕｇ／５０ｕｌ（チューブ）
緩衝系：カコジル酸バッファー（ｐＨ６．５） １００ｍＭ
ＭｎＣｌ２ １０ｍＭ
ＴｒｉｔｏｎＸ−１００ ０．３％
糖供与体：ＵＤＰ−ＧａｌＮＡｃ ０．０８５ｍＭ
Enzyme reaction Substrate: H-type glycolipid (AGL-3) 10 ug / 50 ul (tube)
Buffer system: cacodylate buffer (pH 6.5) 100 mM
MnCl 2 10 mM
TritonX-100 0.3%
Sugar donor: UDP-GalNAc 0.085 mM
ｉｎ ｖｉｔｒｏ合成経路は次のとうりである。
Ｆｕｃα１−２Ｇａ１β１−６Ｍａｎα１−２Ｉｎｓ−Ｐ−Ｃｅｒ＋ＵＤＰ→
Ｈ型糖脂質（ＡＧＬ−３）
Ｇａ１ＮＡｃα１−３（Ｆｕｃα１−２）Ｇａ１β１−６Ｍａｎα１−２Ｉｎｓ−Ｐ−Ｃｅｒ＋ＵＤＰ 合成されるＡ型糖脂質
反応液は５０ｕｌで行う。基質のＡＧＬ−３は先に有機溶媒を除いておく。そのチューブに緩衝液、ＴｒｉｔｏｎＸ−１００、ＭｎＣｌ２、ＵＤＰ−ＧａｌＮＡｃを加え最後に酵素液を加え３７℃、３時間反応を行う。
The in vitro synthesis route is as follows.
Fucα1-2Ga1β1-6Manα1-2Ins-P-Cer + UDP →
H-type glycolipid (AGL-3)
Ga1NAcα1-3 (Fucα1-2) Ga1β1-6Manα1-2Ins-P-Cer + UDP A-type glycolipid to be synthesized The reaction solution is 50 ul. The organic solvent is previously removed from the substrate AGL-3. A buffer solution, Triton X-100, MnCl 2 and UDP-GalNAc are added to the tube, and finally an enzyme solution is added, followed by reaction at 37 ° C. for 3 hours.
Ｃ１８逆層クロマトカートリッジを用いた反応産物の精製
上記の酵素反応でＨ型糖脂質（ＡＧＬ−３）を基質にして糖転移酵素反応により、もう一方の基質であるＵＤＰ−ＧａｌＮＡｃからＮ−アセチルガラクトサミン（ＧａｌＮＡｃ）がＡＧＬ−３に移りＡ型糖脂質がｉｎ ｖｉｔｒｏで生合成される。
Purification of reaction product using C18 reverse-layer chromatographic cartridge N-acetylgalactosamine from UDP-GalNAc, which is the other substrate, by glycosyltransferase reaction using H-type glycolipid (AGL-3) as a substrate in the above enzyme reaction (GalNAc) moves to AGL-3 and A-type glycolipid is biosynthesized in vitro.
Ａ型糖転移酵素として、α−Ｎ アセチルガラクトサミン転移酵素を使用している。 As the A-type glycosyltransferase, α-N acetylgalactosamine transferase is used.
反応生成物のＡ型糖脂質は基質（ＡＧＬ−３）に比べて、非常に微量であるため酵素反応終了後、反応液から更に脂質を精製する。 Since the type A glycolipid of the reaction product is much smaller than the substrate (AGL-3), the lipid is further purified from the reaction solution after completion of the enzyme reaction.
終了後反応チューブへ１ｍｌの水を加え懸濁し、溶液すべてを予め平衡化していたＣ１８逆層クロマトカートリッジにのせる。自然落下で溶出液をチューブに分取する。
更にもう１ｍｌの水を加え、カートリッジを十分洗い込む。
After completion, 1 ml of water is added to the reaction tube to suspend it, and the whole solution is placed on a C18 reverse layer chromatography cartridge that has been equilibrated in advance. Dispense the eluate into tubes by natural fall.
Add another 1 ml of water and wash the cartridge thoroughly.
次に、メタノール２ｍｌ、クロロホルム／メタノール、２／１混液２ｍｌを次々に流し、脂質をカートリッジから溶出し、別のガラスチューブで分取する。
Ｃ１８逆層クロマトカートリッジでは水系で過剰の酵素蛋白質、ＵＤＰ−ＧａｌＮＡｃが洗い出され、最後に有機溶媒（クロロホルム／メタノール混液）で脂質がカートリッジから溶出する。
逆層の高速液体クロマトグラフィーの原理と同じである。
Next, 2 ml of methanol, 2 ml of chloroform / methanol, and 2/1 mixture are flowed one after another, and the lipid is eluted from the cartridge and separated by another glass tube.
In the C18 reverse layer chromatography cartridge, excess enzyme protein and UDP-GalNAc are washed out in an aqueous system, and finally lipid is eluted from the cartridge with an organic solvent (chloroform / methanol mixture).
This is the same as the principle of reverse layer high performance liquid chromatography.
薄層クロマトグラフィーとＴＬＣ免疫染色法
カートリッジからから溶出された有機溶媒に脂質が存在するので、チューブの溶媒をエアーポンプで飛ばし、少量の溶媒（５０ｕｌぐらい）で脂質を溶かし、通常の方法で展開溶媒（Ｃ／Ｍ／Ｗ：６０／３５／８）を用いてＴＬＣで脂質を展開する。
Thin layer chromatography and TLC immunostaining Since lipid is present in the organic solvent eluted from the cartridge, the solvent in the tube is blown off with an air pump, and the lipid is dissolved with a small amount of solvent (about 50 ul). Lipid is developed by TLC using a solvent (C / M / W: 60/35/8).
その後、抗Ａ型抗体（モノクローナル抗体）を用いて免疫染色し、生じたＡ型糖脂質を検出する。発色には酵素抗体法（コニカイムノステイン法）あるいはＥＬＣ法を使用する。
通常これらの反応は放射性同位体（１４Ｃ、ＵＤＰ−ＧａｌＮＡｃ）を用いて、反応後、薄層クロマトグラフィーに展開後、Ｘ線のフィルムの感光によるオートラジオグラフィー法で検出する方法が使われているが、感度が問題にならなければ別の方法を使うことができる。
Thereafter, immunostaining is performed using an anti-type A antibody (monoclonal antibody), and the resulting type A glycolipid is detected. Enzyme antibody method (Konica immunostain method) or ELC method is used for color development.
Usually, these reactions are performed using a radioisotope ( 14 C, UDP-GalNAc), after reaction, developed in thin-layer chromatography, and then detected by autoradiography using X-ray film exposure. However, if sensitivity is not an issue, you can use another method.
未反応のＨ型糖脂質もこの系の中に存在するが、同じ脂質なので薄層クロマトグラフィーでは分離できるので問題ない。使う抗体は抗Ａ型抗体（モノクローナル抗体）なので、他のＨ型糖脂質のような糖脂質とは交叉反応しない。検出感度の問題である。酵素活性が高ければ確実に検出できる。 Unreacted H-type glycolipid is also present in this system, but since it is the same lipid, it can be separated by thin layer chromatography, so there is no problem. Since the antibody used is an anti-A antibody (monoclonal antibody), it does not cross-react with other glycolipids such as H-type glycolipids. It is a problem of detection sensitivity. If the enzyme activity is high, it can be reliably detected.
この結果、レーン１はＡ型赤血球膜より調整した中性糖脂質画分を薄層クロマトグラフィーにのせた。右の抗体による反応では抗Ａ型抗体で反応する主要な糖脂質が上からＡａ、Ａｂ、Ａｃバンドが確認できた。
As a result, in
これに対し、ヒト細胞株より調整した酵素画分を用いて反応させたレーン２では酵素を加えないレーン３の反応に比べて免疫染色で検出された。そして、そのバンドはＡ型糖脂質のバンドが基質であるキノコ由来のＨ型糖脂質の下に検出され、構造としては図7の構造であると考えられる。
類似のヒト細胞株でも同様な結果が得られた。
In contrast,
Similar results were obtained with similar human cell lines.
ブナシメジの酸性糖脂質画分には人血液型類似糖脂質、Ｈ型糖脂質、Ｂ型糖脂質がかなり多く存在する。これらの正確な構造が決定された。
抗体との反応性も殆ど差はなく、今までの血液型判定用には赤血球そのものを抗体と直接反応させていたが、ブナシメジ由来の糖脂質を用いれば代替え品として、使用できる。
The acidic glycolipid fraction of Bunashimeji contains a large amount of human blood type-like glycolipids, H-type glycolipids, and B-type glycolipids. Their exact structure has been determined.
There is almost no difference in reactivity with the antibody, and until now the erythrocyte itself has been directly reacted with the antibody for blood type determination, but it can be used as an alternative if glycolipid derived from Bunashimeji is used.
更に実用化のためには血液型の判定や輸血の際には表試験、裏試験のクロスマッチが必要であるが、キノコ由来の糖脂質を用い、しかも構造既知のＨ型糖脂質の前駆体を用いてｉｎ ｖｉｔｒｏでＡ型糖脂質を合成できる方法を確立できたことは、安価な血液型類似糖脂質を供給できる。 In addition, for practical use, cross-match between front test and back test is necessary for blood type determination and blood transfusion, but it uses mushroom-derived glycolipids and is a precursor of H-type glycolipids with known structure The fact that a method capable of synthesizing A-type glycolipids in vitro using sucrose has been established, can provide inexpensive blood group-like glycolipids.
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