JPH01216267A - Enzyme immunoassay method for hd antigen - Google Patents
Enzyme immunoassay method for hd antigenInfo
- Publication number
- JPH01216267A JPH01216267A JP4075188A JP4075188A JPH01216267A JP H01216267 A JPH01216267 A JP H01216267A JP 4075188 A JP4075188 A JP 4075188A JP 4075188 A JP4075188 A JP 4075188A JP H01216267 A JPH01216267 A JP H01216267A
- Authority
- JP
- Japan
- Prior art keywords
- antigen
- antibody
- bovine
- neugc
- small intestine
- 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.)
- Pending
Links
- 239000000427 antigen Substances 0.000 title claims abstract description 128
- 102000036639 antigens Human genes 0.000 title claims abstract description 124
- 108091007433 antigens Proteins 0.000 title claims abstract description 124
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 30
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 30
- 238000003018 immunoassay Methods 0.000 title claims description 8
- 241000283690 Bos taurus Species 0.000 claims abstract description 53
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims abstract description 26
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 210000000813 small intestine Anatomy 0.000 claims description 47
- 239000012085 test solution Substances 0.000 claims description 9
- FDJKUWYYUZCUJX-KVNVFURPSA-N N-glycolylneuraminic acid Chemical compound OC[C@H](O)[C@H](O)[C@@H]1O[C@](O)(C(O)=O)C[C@H](O)[C@H]1NC(=O)CO FDJKUWYYUZCUJX-KVNVFURPSA-N 0.000 abstract description 40
- 239000007788 liquid Substances 0.000 abstract description 38
- 150000002270 gangliosides Chemical class 0.000 abstract description 19
- 239000000203 mixture Substances 0.000 abstract description 12
- 210000004369 blood Anatomy 0.000 abstract description 9
- 239000008280 blood Substances 0.000 abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 9
- 150000002632 lipids Chemical class 0.000 abstract description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 abstract description 6
- 241000283073 Equus caballus Species 0.000 abstract description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 abstract description 4
- 108010071390 Serum Albumin Proteins 0.000 abstract description 2
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- 238000000034 method Methods 0.000 description 39
- 108090000288 Glycoproteins Proteins 0.000 description 34
- 102000003886 Glycoproteins Human genes 0.000 description 34
- 239000000243 solution Substances 0.000 description 29
- 238000002835 absorbance Methods 0.000 description 27
- 241000287828 Gallus gallus Species 0.000 description 26
- 235000013330 chicken meat Nutrition 0.000 description 24
- 239000012888 bovine serum Substances 0.000 description 23
- 238000005259 measurement Methods 0.000 description 22
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 17
- 238000002360 preparation method Methods 0.000 description 16
- 239000000872 buffer Substances 0.000 description 14
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- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 6
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- SUHQNCLNRUAGOO-UHFFFAOYSA-N N-glycoloyl-neuraminic acid Natural products OCC(O)C(O)C(O)C(NC(=O)CO)C(O)CC(=O)C(O)=O SUHQNCLNRUAGOO-UHFFFAOYSA-N 0.000 description 5
- FDJKUWYYUZCUJX-UHFFFAOYSA-N N-glycolyl-beta-neuraminic acid Natural products OCC(O)C(O)C1OC(O)(C(O)=O)CC(O)C1NC(=O)CO FDJKUWYYUZCUJX-UHFFFAOYSA-N 0.000 description 5
- 238000004040 coloring Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 244000309466 calf Species 0.000 description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 4
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 4
- 235000019797 dipotassium phosphate Nutrition 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
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- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
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- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
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- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 description 2
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- 241000282412 Homo Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 230000004520 agglutination Effects 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- MGWWWSRHCOVLIU-UHFFFAOYSA-N benzene-1,3-diol;hydrochloride Chemical compound Cl.OC1=CC=CC(O)=C1 MGWWWSRHCOVLIU-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 2
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- 235000020958 biotin Nutrition 0.000 description 2
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- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 description 2
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
Description
［産業上の利用分野］
本発明は、アルカリフォスファターゼ楳識ＨＤ抗原に関
するもので、主として臨床的診断におい“ てＨＤ抗
原の検出・測定等に役立てるものである。[Industrial Application Field] The present invention relates to alkaline phosphatase recognition of HD antigens, and is mainly useful for detecting and measuring HD antigens in clinical diagnosis.
【従来の技術］
現在、酵素免疫測定法（以下、ＥＩＡ法、と記す）は、
高感度であり、特殊な機器を必要としないため、次第に
普及しつつある。ＥＩＡ法による抗原の測定法には、大
きく分けて二つの方法がある。
それは、競合法と非競合法とである。しかし、両者には
一長一短がある。後者、即ち非競合法では固相化した抗
体と酵素標識抗体を用いる測定法（いわゆるサンドイッ
チＥＩＡ法）が−船釣である。
このサンドイッチＥＩＡ法は抗体に８！識する酵素及び
この酵素の基質等を適当に選択することにより、放射免
疫測定法（ＲＩＡ法）に匹敵するが、それ以上の感度を
得ることが可能である。ところが、この′サンドイッチ
ＥＩＡ法では、原理的に被検物質である抗原分子（又は
抗原粒子）が抗体との結合部位を二カ所以上持つ場合以
外適用できない、これに対し、競合法ではサンドイッチ
ＥＩＡ法はどの感度は期待できないが、被検物質である
抗原分子（又は抗原粒子）が抗体との結合部位を１カ所
しか持っていない場合にも適用できる。
ところで、近年腫瘍関連抗原としてＨＤ抗原（Ｈａｎｇ
ａｎｕｔｚｉｕ−Ｄｅｕｃｈｅｒ ’ＡｎＬｉｇｅｎｓ
）が注目されている。 ＨＤ抗原とは、Ｎ−グリコリル
ノイラミン酸（ＮｅｕＧｃ ）残基をエピトープとして
含む複合糖鎖を示し、糖蛋白質又はガングリオシドとし
て存在する。これに対しＨＤ抗体とは、Ｎ−グリコリル
ノイラミン酸含有糖鎖と、Ｎ−グリコリルノイラミン酸
残基を必須用件として反応する抗体を言う、ＨＤ抗原は
、動物界に広く分布する異好性抗原の一種でヒト及びニ
ワトリには通常存在しないと考えられてきた。このＨＤ
抗原がヒト及びニワトリのＭ瘍組紘に存在することが明
らかにされ〔東秀好他、脂質生化学研究、 Ｖｏｌ、２
６． ｐ、９２〜９５，１９８４ ） （Ｈ。
Ｈｉｇａｓｈｉ ｅｔ ａｌ、、 Ｊ、 ｏｆ Ｂｉｏ
ｅｌ＋ｅｍｉｓｔｒｙ （Ｔｏｋｙｏ）ｗ Ｖｏｌ、
９５． ｐ、 ７８５〜７９４ 、１９８４ ）、ＨＤ
抗原の高感度がっ筒便な測定法の開発が望まれている。
ＨＤ抗原の測定法としては、ＨＤＤ原ガングリオシドを
抽出・部分精製してＷｉ１！Ｉクロマトグラフィー上に
展開しｖｌ、素免疫染色する方法（Ｈ，Ｈｉｇａｓｈｉ
ｅＬ ａｌ、、 Ｊ、 Ｂｉｏｃｈｅｍｉｓｔｒｙ （
Ｔｏｋｙｏ）＊ Ｖｏｌ、 ９５ｗ ｐ、 １５１７〜
１５２０、 １９８４ ）があるが、これはＨＤ抗原〃
ングリオシドの測定に対しては有効であるが、糖蛋白性
のＨＤ抗原には、適用できない上、提作が繁雑で多量の
検体を扱うには適当でない、この他、血清ＨＤ抗原の測
定を試みた例として、森藤らの報告〔森藤隆夫他、肝臓
＊ Ｖｏｌ、２８１ ｐ、３２４〜３２９゜１９８７
）がある、この方法は、虫ず試料血清を希釈し、１００
°Ｃ３０分間加熱した後、これを遠心して上清を得る。
この血清の加熱遠心上清に、ＨＤ抗抗体強性性血清加え
、室温で２時間反応させる０次に、この反応液をあらか
じめＨＤ抗原を結合させた９６六マイクロプレートに加
えて反応させ洗浄の後、アルカリフォスファターゼ楳識
抗ヒ） ＩｇＧ又はアルカリ７オス７７ターゼａｔ抗ヒ
トＩｇＭを加えで反応させ、再び洗浄の後アルカリフォ
スファターゼ基質液を加えて反応し、吸光度を測定する
。試料血清の加熱遠心上清が入らない場合の吸光度と比
較して、試料血清の加熱遠心上清が入った場合の吸光度
の減少分を測定することにより、試料血清中のＨＤ抗原
を測定する方法である。この方法では、試料血清を加熱
処理しているため、試料血清中のＨＤ抗体及びその他の
蛋白質に由来する非特異反応の１１７を受は難い、その
反面、試料血清中の大部分の蛋白質は加熱処理により不
溶化しでしまうため、ＨＤ抗原糖蛋白質の内はんの一部
しか測定し得ない上、ＨＤＤ体陽性血清を大量に継続的
に得ることは現実的に難しい。
ＧＭ＊（ＮｅｕＧｃ） Ｗングリオシド（木下記構造）
を二＊ＧＭ３（ＮｅｕＧｃ）ガングリオシドＮｅｕＧｃ
（２２−＋３Ｇａｌβ１−＋４ＧＩｃβ１→ｃｅｒＮｅ
ｕＧｃα：ａ−Ｎ−グリコリルノイラミン酸Ｇａｌβ
：β−〃ラクトース
Ｇｌｃβ ：β−グルコース
Ｃｅｒ ：セラミド
ワトリに免疫してＨＤＤ原測定に有効なニワトリＨＤ抗
体が得られることはすでに報告されている（ Ｙ、 Ｆ
ｕｊｉ ｅｔ ａｌ、、 Ｍｏ１ｅｃｕｌａｒ Ｉｍｍ
ｕｎｏｌｏｇｙ、 Ｖｏｌ、 １９ｓ Ｎｏ、 Ｌｐ、
８７〜９４．１９８２ ） 、ウサギのポリクローナル
抗体（Ｒ，Ａ、 Ｌａ１ｎｅ ｅｔ ａｌ、、 Ｊ、
Ｂｉｏｌ、 Ｃｈｅｗ、１Ｖｏｌ、 ２４９゜ｐ、 ４
４６０〜４４６６、１９７４）、マウスモノクローナル
抗体〔山崎＋Ｉ１１彦他、脂質生化学研究、 Ｖｏｌ、
２９゜ｐ、２５〜２８．１９８７ ）、ヒトモノクロ
ーナル抗体（薄場修他９日本癌学会総会記事、第４６回
、ｐ。
３０４、演題番号１１９６．１９８７）等の報告もある
。
従来の酵素免疫測定法に用いられている酵素標識抗原は
、抗原と酵素を化学的に結合させる例が大部分である。
この方法では抗原又は酵素を化学的に処理する必要があ
り、その際抗原又は酵素に損傷を与える危険がつきまと
う、その他遺伝子操作により酵素標識抗原を作成した例
（Ａ、 Ｐｅｔｅｒｈａｎｓ＊ｅｔａ１．＊ Ａｎａｌ
、 Ｂｉｏｅｈｅｍ、 Ｖｏｌ、１６３ｙ ｐ、４７０
〜４７５＊ １９８７）もあるが、高度な技術と特殊な
設備を必用とする上、現技術水準では、遺伝子繰作によ
り糖鎖を変えることは不可能である。即ち、この方法で
は酵素標ｆｉＨＤ抗原は得られない、糖蛋白質へＮ・グ
リコリルノイラミン酸残基、即ちＨＤ抗原を酵素的に導
入する試みは、ボールソンらのグループが行なっており
（ＨｏＨｏＨｉｇａ ＆ Ｊ、Ｃ０Ｐａｕｌｓｏｎ＊
Ｊ、 Ｂｉｏｌ。
Ｃｈｅｔａ、＊ Ｖｏｌ、２６０． ｐ、８８３８−８
８４９．１９８５ ）、原理的には、任意の酵素にＨＤ
抗原を酵素的に導入し、酵素標ｆｆ１ＨＤ抗原を調製す
ることが可能であるが、現実には什われていない、又、
ＨＤ抗原に限らず、酵素が元米持っている抗原性を利用
し、これを酵素標識抗原として用いる酵素免疫測定法は
、従来存在しなかったのである。
ところで動物生体内の蛋白質は、その多くが糖蛋白質で
あり、その内の多くが、シアル＃（Ｎ−７セチルノイラ
ミン酸及びＮ−グリコリルノイラミン酸誘導体）残基を
持つている。従って、ヒト及びニワトリを除く他の動物
白米の酵素には、多かれ少なかれＨＤ抗原を含有する、
即ち元来酵素？”ａＨＤＨＤ抗原得る可能性を持ってい
る。
ウシ小腸由来アルカリ７ｔス７アターゼは、−分子あた
りの比活性が高く、比較的安定性が良く安価である等の
理由により、抗体又は抗原の標識化酵素として広く用い
られている。このアルカリ７オスフアターゼには従来、
シアル酸（Ｎ−７セチルノイラミン酸及びＮ−グリコリ
ルノイラミン酸誘導体）残基は含まれでいないとされて
きた〔ＨｏＮ、 Ｆｅｒｎｌｅｙ、 Ｔｈｅ Ｅ
ｎｚｙｍｅｓ Ｖｏｌ、■ （３ｒｄ ｅｄｉｔｉ
ｏｎ）ｗ ＡｃａｄｅｍｉｃＰｒｅｓｓ＋ １９７Ｌ
ｐ、４２４）、即ち、未修飾のウシ小腸由来アルカリ
７ｔス７アターゼが、ＨＤ抗体と強い親和性を持つこと
は、今まで誰にも推測し得なかったわけである。
［発明が解決しようとする問題点］
本発明者らは、前記従来技術の問題点に対して、種々検
討を行った結果、ウシ小腸由来アルカリ７ｔス７アター
ゼにはＨＤ抗原はないと考えられていたにもかかわらず
、ＨＤ抗原が存在することを見出し、ウシ小腸由来アル
カリ７ｔス７アターゼを、アルカリフォスファターゼＩ
Ｘ識ＨＤ抗原としてｍいることにより、高感度なＨＤ抗
原の酵素免疫測定法を確立し、本発明を完成するに至っ
た。
【問題点を解決するための手段１
本発明は、ウシ小腸由来アルカ１７７オス７７ターゼを
アルカリ７オス７７ターゼ標ａＨＤ抗原として用いる競
合法を原理とした被検液中のＨＤ抗原を測定するための
酵素免疫測定法である。即ち、ウシ小腸白米アルカー１
７オス７アターゼをアルカリフォスファターゼ標ｉａ
ＨＤ抗原として用い、被検液中のＨＤ抗原とＨＤ抗体の
反応が、当該標識ＨＤ抗原とＨＤ抗体との反応を競合的
に阻害することを利用し、かつＨＤ抗体に結合した或い
は結合していないウシ小腸由来アルカリフォスファター
ゼ量を測定することにより、被検液中のＨＤ抗原量を測
定することを特徴とするＨＤ抗原の酵素免疫測定法であ
る。
本発明法をさらに説明すれば、マイクロプレート、ボー
ル、ディスク片又はゲル等の固相に固定化したＨ Ｄ抗
体に、被検液とウシ小腸由来アルカリフォスファターゼ
を、順次或いは、同時に反応させ、固相に結合した（Ｈ
Ｄ抗体に結合した）アルカリフォスファターゼ量或いは
、液相に！ｉｌもしくは残存したアルカリ７オス７７タ
ーゼ量をその酵素活性により測定し、標準ＨＤ抗７ｊＸ
？ｌｌの検量線から、被検液中のＨＤ抗原量を求める。
又、被検液にＨＤ抗体を加え反応させた後或いは、同時
にウシ小腸白米アルカリフォスファターゼを加え、反応
させた後、ＨＤ抗体を固相に集め固相に結合したウシ小
腸由来アルカリフォスファターゼ量或いは、液相に残存
したアルカリフォスファターゼ量を、その酵素活性によ
り測定し、標準ＨＤ抗原液の検量線から、被検液中のＨ
Ｄ抗原量を求める。この際のＨＤ抗体を固相に集める方
法としては、ＨＤ抗体の白米動物のイムノグロブリンに
対する抗イムノグロブリン抗体を固相化して集めるか、
アビジン−ビオチン法（ＨＤ抗体をビオチン化しておき
、アビジンを固相化するが、ＨＤ抗体をアビジン化して
おき固相にビオチンを結合させておくことにより集める
）等の手段がある。
さらに、被検液にＨＤ抗体を加え反応させた後或いは、
同時にウシ小腸由来アルカリフォスファターゼを反応さ
せた後、ＨＤ抗体を凝集させ、遠心して凝集塊と上清に
分離する。その後凝集塊又は上清のウシ小腸白米アルカ
リフォスファターゼ量を、その酵素活性により測定し、
標準ＨＤ抗原液の検量線から、被検液中のＨＤ抗原量を
求める。
この際のＨＤ抗体を凝集させる方法としては、ＨＤ抗体
の白米動物のイム／グロブリンに対する抗イムノグロブ
リン抗体を加えて凝集させるか、アビジン−ビオチン法
（ＨＤ抗体をビオチン化しておき、アビジンを単独で或
いは重合させて、或いは、他の蛋白質等に結合させて加
え凝集させるか、ＨＤ抗体を７ビジン化しておき、ビオ
チン化蛋白質等ビオチンを多価とした物質を加え凝集さ
せる）等の手段がある。
本発明のウシ小腸白米アルカＩ７７オス７アターゼは、
ウシ小腸より抽出・精製して使用するか、市販されてい
るものを購入し、必要があればさらに精製して使用すれ
ばよい。
又、ウシ小腸由来アルカリフォスファターゼの他に、ウ
シ腎臓由来、ラット肝臓由来、ラット小腸白米、ブタ腎
臓由米等各動物臓器由来のアルカリ７オス７７ターゼを
用いて７ルカリ７オス７７ターゼ標識ＨＤ抗原としてＨ
Ｄ抗原の測定を行うことも可能である。
本発明のＨＤ抗体は、ＨＤ抗原（Ｎ−グリコリルノイラ
ミン酸を含む糖蛋白質又はガングリオシド）を、ニワト
リ又は他の動物（＠乳類又はＳ類）に免疫することによ
り得られる抗血清、ヒ）ＨＤ抗抗体強性性血清はヒト或
いは、マウスのモノクローナル抗体を必要に応じて精製
して使用することができる。
【作用】
本発明により、抗原と酵素を結合させるという繁雑な操
作なしに高感度かつ簡便なＨＤ抗原の酵素免疫測定法が
確立される。
以下に、本発明を実施例により説明するが、本発明はこ
れにより何ら限定されるものではない。
参考例
ウシ小腸由来アルカリ７オスフアターゼとＨＤ抗体の親
和力の測定
（１） ＧＭｓ（ＮｅｕＧｃ） ｌｆングリオシドの調
製ＥＤＴＡＤ加ウマ（サラブレッド）血液１０１より赤
血球を分離し、１％酢酸水溶液により溶血させてウマ赤
血球膜ゴーストを得た。このウマ赤血球膜ゴーストより
、クロロホルム・メタノール混液により総脂質画分を抽
出し、アセトン沈澱、ＤＥＡＥ−セフ７デツクスＡ−２
５カラムクロマトグラフイー、斉藤・箱守らの方法（Ｔ
、 ５ａｉｔｏｓ Ｓ。
Ｈａｋｏｅｏｒｉ＊ Ｊ、 ｏｆ Ｌｉｐｉｄ Ｒｅｓ
、＊ Ｖｏｌ、１’Ｌ ｐ、２５’Ｌ １９７１ ）に
よりＧＭｓ（ＮｅｕＧｃ） Ｉｆングリオシド（車下記
構造）を精製し、標品的０．７２．を得た。
本ＧＭ３（ＮｅｕＧｃ）ｆｆングリオシドＮｅｕ Ｇｅ
６２−ｅ ３ Ｇａ ｌβ１−＋４ＧＩｃβ１−ｅｃ
ｅｒＮｅｕＧｃ７：ｆｆ−Ｎ−グリコリルノイラミン酸
Ｇａｌβ ：β−〃ラクトース
Ｇｌｃβ ：β−グルコース
Ｃｅｒ ：セラミド
（２）ニットＩＪＨＤ抗体の調製
前記（１）で得たＧＭａ（ＮｅｕＧｃ） Ｎングリオシ
ド約１ ｘｇ、ニワトリ血清アルブミン分［にワトリ血
清５０％飽和硫安上清７５％飽和硫安沈ｍ画分）約１
ｍｇに、精製水０．５ｍ１．７０インド・コンプリート
・アジュバント（デイ７コ社製）０．５ｉ／を加えて乳
化し、ニワトリ（ブロイラー、畔化３０日目）の腹部に
注射し免疫した。以後２週問おきに５回、前記と同様な
方法で免疫し１４週目間採血した。
採血した血液は、３７℃で１時間保温し凝固させた後、
遠心して抗血清を得た。この抗血清に１７２倍量の飽和
硫安溶液を加えて抗体成分を塩析させ溶解、透析の後、
ＤＥＡＥ−セルロースカラムクロマトグラフィーにより
精製した。このようにして得たニワトリｒｇａ画分を、
ＧＭｓ（ＮｅｕＧｃ） Ｗングリオシドを疎水的に結合
したオクチルセフ７０−ス４Ｂカラム（Ｙ、Ｈｉｒａｂ
ａｙａｓｈｉ ｅｔ ａｌ、、 Ｊ、 Ｂｉｏｃｈｅｍ
ｉｓｔｒｙ（Ｔｏｋｙｏ） Ｖｏｌ、９４． ｐ、３２
７ ＝ ３３０．１９８３参照）にかけて、ニワトリ抗
ＧＭ３（ＮｅｕＧｃ）抗体（即ちニワトリＨＤ抗体）を
得た。
（３）ウシ小腸由来アルカリフォスファターゼとニワ）
’ＪＨＤ抗体との親和力の測定
前記（２）で得たニワトリＨＤ抗体を０．０５％ＮａＮ
＝−ＰＢＳ （塩化ナトリウム８．０ｇ、塩化カリウム
０．２０ｇ、リン酸＝水素二ナトリウム・１２水塩２，
８８．、リン酸二水素−カリウム０．２ ｏ、、を精製
水で溶解して１．０１として得た。以後ＰＢＳと記す、
）で、Ａ６．。＝０．０１０となるように希釈し、ポリ
スチレンｇ１９６穴マイクロプレー）Ｓ（住友ベーク２
イト社１）の２４穴に、対照として抗体を含まない０゜
０５％ＮａＮ３− Ｐ Ｂ Ｓを別の２４穴に各式５Ｑ
ｔｔｌずつ加えて室温で一夜放置し、抗体をプレートの
各穴に結合させた（対照とした穴には抗体の結合はない
）このプレートの各穴中の液を吸引除去した後、０．０
５％Ｔｗｅｅｎ２０−ＰＢＳ３００〜４００μｌにて各
式を３回洗浄し、各穴中の液を充分除去し、１％ＣＯＡ
（ニワトリ卵白アルブミン、５回結晶（生化学工業社
製）、以後ＣＯＡと記す、）−０゜０５％ＮａＮ５−
Ｐ Ｂ Ｓ ３００１１１を各式に加え３７℃で３時間
放置した。このプレートの各穴中の液を吸引除去した後
、０．０５％Ｔｗｅｅｎ２０−Ｐ Ｂ Ｓ ３００〜４
００μｌにて各式を３回洗浄し各穴中の液を充分除去し
た。ニワ）ＩＪＨＤ抗体を結合した穴の２穴ずつと対照
穴の２穴ずつの合計４穴ずつに、ウシ小腸由来アルカリ
７オス７Ｔターゼ（Ｐｈｏｓｐｈａｔａｓｅａｌｋａｌ
ｉｎｅ ｆｒｏｍ ｃａｌｆ １ｎｔｅｓｔｉ
ｎｅ ｆｏｒ ｅｎｚｙｍｅ ｉｗｕｎｏａｓｓ
ａｙ（３ｘｇ１０，３ｘ１）５６７７４４ Ｌｏｔ、１
０６７５３１−５７１０ｃｔ、８７（ベーリン〃−・マ
ンハイム・山之内社！Ｉ！り）を１７４００から１／４
００ Ｘ ２−”まで１％ＣＯＡ−０，７％ＮａＣ１−
１ｚＭＭｇＣｉ’ｚ −３０ｘＭ ）リエタノールアミ
ンａｍ液（ｐ夏］＝７．６）にて倍々希釈した液４０μ
ｌずつ加え、３７℃にて２時間反応させた。再び、各穴
中の液を吸引除去し、０．０５％Ｔｗｅｅｎ２０−Ｐ
Ｂ Ｓ ３００〜４００μｌにて各式を７回洗浄し、各
穴中の液を充分除去した後、ＡＬＰローゼ基質液（ジノ
テスト社製）を各式に１００μｌずつ加え３７℃で６０
分間反応させた０次に、ＡＬＰローゼ呈色液（ジノテス
ト社！Ｉりを各式に１００μｌずつ加え波ｆｌ ５００
ｎｍにおける吸光度（対照として６３０ ＋ｍ ）を
マイクロプレートリーダー（ＭＴＰ−３２（コロナ）〕
で測定した。この結果を、第１図に示す。
この実験により、ウシ小腸由来アルカ１７７オスフアタ
ーゼが、ニワトリＨＤ抗体と強い親和性を持つことが明
らかとなった。
比較例Ｉ
ＨＤ抗抗原シングリオシド測定おけるＨＤＤ原化学結合
ペルオキシダーゼとウシ小腸由来アルカリフォスファタ
ーゼの醪素楳識ＨＤ抗原としての比較
（１） ＧＭｚ（ＮｅｕＧｃ） Ｗングリオシドの調製
参考例（１）と同様
このＧＭｓ（ＮｅｕＧｃ） ＷングリオシドをＨＤＤ原
ガングリオシドの代表とする。
（２）ウシ血清ＨＤ抗原糖蛋白質画分の調製ウシ血清１
００１１１に、飽和硫安１００ｚｌを加え室温で３０分
間攪拌し、７０００ｒｐｍ ３０分間遠心〔ＲＡ−７（
クボタ）〕シた後、沈澱物（ウシ血清グロブリン）を除
き、１６２ｉ１の上清を得た。この上清に、１６２ｙｌ
の飽和硫安を加え室温で３０分間攪拌し、７０００ｒｐ
ｍ２時間遠心（ＲＡ−７（クボタ）〕シ、上清を除いて
沈澱物を精製水で溶解した後、０．０５％ＮａＮ＝ −
Ｐ Ｂ Ｓに対して充分透析し、ウシ血清１／２〜３／
４飽和硫安画分６９．０ｉ＋Ｚを得た。
このウシ血清１／２〜３／４飽和硫安画分約６５１１を
、５０ＪＩＭ ）リス塩酸緩衝液（ｐＨ＝ ８．６ ）
に対しで透析した後、２回に分けてＦＰＬＣシステム（
ファルマシア）でＨＤＤ原活性の強い両分を得瓢カラム
はＱ−セフ７０−スカラム（１６Ｘ １１１ａｌＬ１）
を用い、５０ｚＭ ）リス塩酸１１！１ｔ（ｐＨ＝ ８
．６ ）で結合させ、１００ｉ＋Ｍ ＮａＣＺ −５０
ｉ＋Ｍ ）リス塩酸緩衝ｆｉ（ｐＨ＝８．６）から２５
０ｚＭ ＮａＣｌ −５０ｚＭ ）リス塩酸緩衝液（ｐ
Ｈ＝８．６）まで、流速５ｍｊ！／ｗｉｎ ３５分間で
グラシュエンド溶出した。得られたＨＤＤ原活性の強い
画分を集めで、５０ｉ＋Ｍ ）リス塩Ｗ１緩衝１（ｐＨ
＝８．６）に対して透析し、前記と同様な方法でリクク
マトし精製水に対しで透析し、凍結乾燥してウシ血清Ｈ
Ｄ抗原糖蛋白質画分約１５０ｉ＋ｇを得た。得られたウ
シ血清ＨＤ抗原糖蛋白質画分は、シアル酸を約７％含有
する。シアル酸はレゾルシノール−塩酸法〔生化学実験
講座３．脂質の化学１日本生化学会編ｓ １９７４年
ｓ ｐ−８８１により定量した。
（３） ＨＤ抗原化学結合ペルオキシダーゼの調製西洋
ワサビヘルオキシダーゼ（Ｇｒａｄｅ ＬＬｙｏｐｌ＋
１ｌｉｓａｔ＊１０８０９０、ベーリンが−・マンハイ
ム・山之内社製）３．３５ｚｙを０．３ Ｍ Ｎａ Ｈ
ＣＯｓに溶解し、１％２，４−ジニトロフルオロベンゼ
ン懸濁ｖＬ０，１ｍｌを加え室温で１時間反応させた。
０．０６Ｍ Ｎａｌ０＋ ０．５ｇ＋１を加え室温で
３０分間反応の後、０．１６Ｍエチレングリコール０．
５１１１を加え室温で１時間反応させ、１０ｚＭ炭酸ナ
トリウム緩衝液（ｐＨ＝９．５）で４℃−夜透析した。
これに、前記（２）で得たウシ血清ＨＤ抗原糖蛋白質画
分１．６３１ｇ／１０１Ｍ炭酸す） ＋７ウム緩衝液（
ｐＨ−９，５） ０．１ ａｔを加え室温で３時間反応
させ、ＮａＢＣＮＨ，１、７７ｕ／ １０ｚＭ炭酸ナト
リウム緩衝８１（ｐＨ＝ ９．５ ） ０，３ ｘｌを
加え室温でさらに４時間反応させた後、ＰＢＳに対して
透析した。
得られた液の約半量に４倍量の精製水を加え、１０ｍＭ
リン酸水素カリウム緩衝ｔ（ｐＨ＝ ８．０ ）に平衡
化したＤＥＡＥ−セルロースカラム（１６Ｘ８３Ｍ）に
かけ、未反応のペルオキシダーゼを１０ｚＭリン酸水素
カリウム緩衝液（ｐＨ＝８．０）で溶出し、２０ｚＭ、
３０１Ｍ、 ４０１Ｍ、 ５０１Ｍ、 ６０
１Ｍ。
７０ｍＭのリン酸水素カリウム緩衝液（ｐＨ＝８．０）
各々１０１１で順次溶出し、３０〜６０ｉ＋Ｍのリン酸
水素カリウム緩衝液（ｐＨ＝８．０）で溶出された部分
を集めてセントリフ０−ＣＦ−２５（アミコン）にて濃
縮し、ＨＤＤ原化学結合ペルオキシダーゼとした。
（４）ニット１７ ＨＤ抗体の１！４製前記（１）で得
た０Ｍ３（ＮｅｕＧｃ） ｉｆングリオシド約１ ｘｇ
。
ウシ血清アルブミン（コーンＦｒＶ、アーマー社製）１
ｘｇに、精製水０．５ｍｌ１，７０インド・フンブリ
ート・アジュバント（デイ７コ社製）０．５ｉｊ！を加
えて乳化し、ニアトリ（ブロイラー、畔化３０日目）の
腹部に注射し免疫した。以後２週問おきに６回、前記と
同様な方法で免疫し１６週目に採血した。
採血した血液は、参考例（２）と同様の方法でニット１
７ ＨＤ抗体を得た。
（５） ＨＤ抗原化学結合ペルオキシダーゼを用いたＨ
Ｄ抗抗原シングリオシド測定
前記（４）で得たニワトリＨＤ抗体を０．０５％ＮａＮ
３−ＰＢＳで、Ａ□。〜０．０１０にて希釈し、ポリス
チレン製９６穴マイクロプレート（イムノプレート■、
ヌンク社製）の２６穴に、各式５０μｌずつ加えて１０
℃で一夜放置し、ニア）ＩＪＨＤ抗体を各式に結合させ
た。この各穴中の液を吸引除去し、１％Ｃ０Ａ−０，０
５％ＮａＮ＝−Ｐ Ｂ ８３００μｌを加え、３７℃で
２時間放置した後、各穴中の液を吸引除去し、０．０５
％Ｔｗｅｅｎ２０−Ｐ Ｂ Ｓ ３００−４００１ｔｌ
にて５回洗浄し、各穴中の液を充分除去した後、前記（
１）で得た０Ｍ３（ＮｅｕＧｃ） Ｗングリオシド１０
０＃＃／ｍｌ、１０Ｍｇ７ｍ１．１μｇ７ｍｌ 、 １
００ ｎｇ／ＩＩｌ、１０ ｎｇ／ｘｉ、１ ｎｇ／ｍ
ｌ、１００ ｐｇ７ｍｌの各１％Ｃ０Ａ−０，０５％Ｎ
ａＮ＝−ＰＢＳ］！を各々２穴ずつ計１４穴に５０μｌ
ずつ加え、残りの１２穴にはＨＤ抗原を含まない１％Ｃ
０Ａ−０，０５％ＮａＮ５− Ｐ Ｂ Ｓ ５０１１１
を加え３７℃で２時間反応した。各穴中の液を吸引除去
した後、０．０５％Ｔｗｅｅｎ２０−Ｐ Ｂ Ｓ ３０
０〜４００ｕｒにて洗浄し、各穴中の液を充分除去した
。この２６穴にＨＤＤ原化学結合ペルオキシダーゼの１
％ＣＯＡ−ＰＢＳ希釈液４０μｌずつ加え３７℃で２時
間反応させた。各穴中の液を吸引除去した後、０．０５
％Ｔｗｅｅｎ２０−Ｐ Ｂ Ｓ ３００〜４００＃ｊ！
にて７回洗浄し、各穴中の液を充分除去し、ペルオキシ
ダーゼ基質液（０−７ｚニレンシアミン４ ｚｇ、Ｍｅ
Ｉ Ｉｖａｉｎｅ緩衝液（ｐＨ＝ ５．０ ） １０
ｍ１％０．３％Ｈｉｎｔ ０．３３ ｍｌを混和してＩ
ＩＩした〕を各式１００μｌずつ加え３７℃で６０分間
反応した。この２６穴に４ＮＨ２３０，５０μｒずつを
加え、波長４９２ ｎｍにおける吸光度（対照として波
長６３０ ｎｍ ）をマイクロプレートリーグ−（ＭＴ
Ｐ−３２（コロナ）〕で測定した。
ＨＤ抗原を反応させなかった１２穴の平均の吸光度を１
００とした場合のＧＭｓ（ＮｅｕＧｃ） ：Ｉｆングリ
オシドを種々の濃度で反応させた各式の吸光度の百分率
を算出し、第２図に示した。
（６）ウシ小腸白米アルカリフォスファターゼを泪いた
ＨＤＤ原ガングリオシドの測定
前記（４）で得たニット９ＨＤ抗体を０．０５％ＮａＮ
３−ＰＢＳで、Ａ２．。〜０．０１０にて希釈し、ポリ
スチレン製９６穴マイクロプレート（イムノプレート１
、ヌンク社ｇ１）の２６穴に、各式５０μｌずつ加えて
１０℃で一夜放置し、ニワトリＨＤ抗体を各式に結合さ
せた。この各穴中の液を吸引除去し、１％Ｃ０Ａ−０，
０５％ＮａＮ５’ Ｐ Ｂ Ｓ ３００１１１を加え、
３７℃で２時間放置した後、各穴中の液を吸引除去し、
０．０５％Ｔｗｅｅｎ２０−Ｐ Ｂ Ｓ ３００ ＝
４００１ＪＩにて５回洗浄し、各穴中の液を充分除去し
た後、前記（１）で得た０Ｍ３（ＮｅｕＧｃ） Ｉｆン
グリオシド１００／ＪＩＦ７ａ１．１０Ｍｇ／ｗｌ、１
μｇ７ｍｌ、１００ ｎｇ／ｍｌ、１０ ｎｇ／ｘｉ
１１ ｎｙ／ｍｌ 、 １００ ｐｇ／１１の各１％Ｃ
ＯＡ−０，０５％ＮａＮ３−ＰＢＳ溶液を各々２穴ずつ
計１４穴に５０μｌずつ加え、残りの１２穴にはＨＤ抗
原を含まない１％Ｃ０Ａ−０，０５％ＮａＮ５− Ｐ
Ｂ Ｓ ５０μｌを加え３７℃で２時間反応した。各穴
中の液を吸引除去した後、０．０５％Ｔｗｅｅｎ２０−
Ｐ Ｂ Ｓ ３００〜４００１１１１こて洗浄し各穴中
の液を充分除去した。この２６穴にウシ小腸由来アルカ
リ７オス７７ターゼ（Ｐｈｏｓｐｈａｔａｓｅａｌｋａ
ｌ ｉｎｅ ｆｒｏｍ ｃａｌｆ １ｎｔｅｓｔｉ
ｎｅ ｆｏｒ ｅｎｚｙ＋ｅｅ ｉｍ＋ｕｎｏａ
ｓｓａｙ（３ｉ＋ｇ１０，３ｍ１）５６７７４４ Ｌｏ
ｔ、１０６７５３１−５７１０ｃｔ、８７ （ベーリン
〃−・マンハイム・山之内社！Ｉｔ ） ） １／２５
．０００希釈１ ％Ｃ０Ａ−０，７％ＮａＣ１１ｉ＋Ｍ
ＭｙＣｌｚ −３０ｍＭ ） リ エタノールア
ミン緩衝液（ｐＨ＝７．６）溶液４０μｌずつを加え３
７℃で２時間反応させた。各穴中の液を吸引除去した後
、１ｍＭ ＭｇＣ１＊−Ｐ Ｂ Ｓ ３００〜４００μ
ｌにて７回洗浄し、各穴中の液を充分除去し、１ｗＭ
ＭｔｔＣＴｏ−Ａ Ｌ Ｐローゼ基質液（ＡＬＰローゼ
基質液（ジノテスト社製）１００ｍＡ’に、１＊Ｍ Ｍ
ｇＣ１ｚＯ，１０ｍ１’を加えて調製した。〕を各大穴
００μｌずつ加え３７℃で６０分間反応した。この２６
穴にＡＬＰローゼ呈色液（ジノテスト社製）１００μｌ
ずつを加え、波長５００ ｎｍにおける吸光度（対照と
して６３０ ｎｍ ）をマイクロプレートリーダー〔Ｍ
ＴＰ−３２（コロナ）〕で測定した。
ＨＤ抗原を反応させなかった１２穴の平均の吸光度を１
００とした場合のＧＭｓ（ＮｅｕＧｃ） ｉｆガングリ
オシド種々の濃度で反応させた各式の吸光度の百分率を
算出し、第２図に示した。
（７）結果
本実験によりＨＤ抗原ガングリオシドの測定に際し、酵
素標識抗原としてＨＤ抗原化学結合ペルオキシダーゼを
用いるよりも、ウシ小腸由来アルカリ７オス７７ターゼ
を用いる方が数倍高い感度が得られることが明らかとな
り、ウシ小腸由来アルカリフォスファターゼの有用性の
高さが示され瓢
比較例２
ＨＤ抗原糖蛋白質測定におけるＨＤ抗原化学結合ペルオ
キシダーゼとウシ小腸由来アルカリフォスファターゼの
酵素標ＩＨＤ抗原としての比較（１）ウシ血ｔｆｆＨＤ
抗原糖蛋白質画分の調製比較例１−（２）と同様
このウシ血清ＨＤ抗原糖蛋白質画分をＨＤ抗原糖蛋白質
の代表とする。
（２） ＨＤ抗原化学結合ベルオキシグーゼの調製比較
例１−（３）と同様
（３）ニット１７ ＨＤ抗体の調製
比較例１−（４）と同様
（４） ＨＤ抗原化学結合５ペルオキシダーゼを用いた
ＨＤ抗原糖蛋白質の測定
比較例１−（５）のＧＭ、（ＮｅｕＧｃ）ガングリオシ
ドをウシ血清ＨＤ抗原糖蛋白質画分に替えて同様の測定
を行なった。結果は、第３図に示した。
（５）ウシ小腸由来アルカリ７オス７７ターゼを用いた
ＨＤ抗原糖蛋白質の測定
比較例１−（６）のＧＭ、（ＮｅすＧｃ） ：／ｆガン
グリオシドウシ血清ＨＤ抗原糖蛋白質画分に替えて同様
の測定を行なった。結果は、第３図に示した。
（６）結果
本実験によりＨＤ抗原糖蛋白質の測定に際し、酵素標識
抗原としてＨＤ抗原化学結合ベルオキシグーゼを用いる
よりも、ウシ小腸由来アルカリフォスファターゼを用い
る方が数倍高い感度が得られることが明らかとなり、ウ
シ小腸由来アルカリフォスファターゼの有用性の高さが
示されｐ、 □実施例１
ウシ小腸由来フルカ１ノアオス７７ターゼを用いたＨＤ
抗ａｘｙングリオシドの測定（二段階法）（１） ＧＭ
ａ（ＮｅｕＧｃ） ｉｆガングリオシド調製参考例（１
）と同様
このＧＭｚ（ＮｅｕＧｃ） ＩｆガングリオシドＨＤ抗
原ガングリオシドの代表とする。
（２）ニットＩ） ＨＤ抗体のＷ４！！参−寄倒（２）
と同様
（３）ウシ小腸白米アルカ１７７オス７７ターゼを用い
たＨＤ抗原の測定
ニット９ ＨＤ抗体を０．０５％ＮａＮ３− Ｐ Ｂ
Ｓで、Ａ、。＝ｏ、ｏｏｓとなるように希釈し、ポリス
チレン製９６穴マイクロプレート（イムノプレート１１
ヌンク社製）の２７穴に、各式５０μｌずつ加えて４℃
で３日間放置し、ニワトリＨＤ抗体を各式に結合させた
。このプレートの各穴中の液を吸引除去した後、０．０
５％Ｔｗｅｅｎ２０−Ｐ Ｂ Ｓ ３００〜４００μｌ
にで各式を３回洗浄し、各穴中の液を充分に除去し、１
％ＣＯＡ 〜０．０５％ＮａＮ５− Ｐ Ｂ Ｓ ３０
０μｌを各式に加え、３７℃で２時間放置した。このプ
レートの各穴中の液を吸引除去した後、各式を０．０５
％Ｔｗｅｅｎ２０−Ｐ Ｂ Ｓ ３００〜４００１１１
にて３回洗浄し、各穴中の液を充分除去した。その２０
穴に前記（１）で得たＧＭｓ（ＮｅｕＧｃ） Ｉｆング
リオシドを１μｇ７ｍｌからＩ Ｘ ２−’μｇ７ｍｌ
まで１％Ｃ０Ａ−０，０５％ＮａＮ３− Ｐ Ｂ Ｓに
で倍々希釈し、２穴ずつに５Ｏｔｔｌずつ加えた。残り
の７穴には、１％Ｃ０Ａ−０，０５％ＮａＮ５− Ｐ
Ｂ Ｓ ５０μｒずつを加えて３７℃で２時間反応した
。このプレートの各穴中の液を吸引除去した後、０．０
５％Ｔｗｅｅｎ２０−Ｐ Ｂ Ｓ ３００−４００μｌ
で７回洗浄し各穴中の液を充分除去し、ウシ小腸白米ア
ルカリ７ｔス７アターゼ（ｐｈｏｓｐｈａｔａｓｅａｌ
ｋａｌｉｎｅ ｆｒｏｍ ｃａｌｆ １ｎｔｅｓｔｉｎ
ｅ ｆｏｒ ｅｎｚｙｒｌｌｅｉａｍｕｎｏａｓｓａｙ
（３ｍｇ７０．３ｍ１）５６７７４４ Ｌｏｔ、１０６
７５３１−５７７Ｏｃｔ、８７（ベーリン〃−・マンハ
イム・山之内社製）〕を１１％Ｃ０Ａ−０７％ＮａＣ１
１％Ｍ ＭｇＣｂ−３０％Ｍ ）リエタ／−ルアミンｌ
ｔｌ衝液（ｐＨ＝７．６）にて１／１２，５００に希釈
した液４０μｌずつを各式に加え３７℃で３時間反応さ
せた。再び、各穴中の液を吸引除去した後、１％ＭＭｇ
ｃＴｏ −０，０５％Ｔｗｅｅｎ２０−ＰＢＳ３００＝
４００μｌで７回洗浄し、各穴中の液を充分除去して、
ＡＬＰローゼ基質液（ジノテスト社製）を各式に１００
μｌずつ加え３７℃で６０分間反応させた０次にＡＬＰ
ローゼ呈色液（ジノテスト社製）を各式に１００μｌず
つ加え、波５．５００ ｒｍにおける吸光度（対照とし
で６３０ ｒｍ ）をマイクロプレートリーダー（ＭＴ
Ｐ−３２（コロナ）〕で測定した。
ＨＤ抗原を添加しない７穴の平均の吸光度を１００とし
た場合のＧＭ、（ＮｅｕＧｃ）ガングリオシドを種々の
濃度で加えた各式の吸光度の百分率を算出し、第４図に
示した。
（４）発明の効果
この実験により、ニワトＩＪＨＤ抗体に対するウシ小腸
白米アルカ１７７オスフアターゼの反応をＨＤ抗抗原シ
ングリオシド阻害することが明らかとなり、この現象を
利用してＨＤ抗ｒＭｆｆングリオシドを高感度に測定す
ることが可能であることが明らかとなった。
実施例２
ウシ小腸由来アルカ１７７オス７７ターゼを用いたＨＤ
抗原糖蛋白質の測定（二段階法）（１）ウシ血清１７２
〜３７４飽和硫安画分の調製ウシ血清１００ｉ１’に、
飽和硫安１００ｍｍ！を加え室温で３０分間攪拌し、７
０００ｒｐｍ ３０分間遠心〔ＲＡ−７（クボタ〕〕シ
た後、沈澱物を除き１６２ ｍｌの上清を得た。この上
清に、１６２Ｒ１の飽和硫安を加え室温で３０分間攪拌
し、７０００ｒｐｍ２時間遠心（ＲＡ−７（クボタ）〕
シ沈澱物を精製水で溶解した後、０．０５％ＮａＮ５−
Ｐ Ｂ Ｓに対して充分透析しウシ血清１／２〜３／
４飽和硫安画分６９．０１１１を得た。得られたウシ血
清１／２〜３／４飽和硫安画分の０．０５％ＮａＮａ
−Ｐ Ｂ Ｓ中での２８０ ｎｍにおける吸光度は３４
．５で、シアル酸は１．７８μｍｏｌｅ７ｍｌであった
。〔シアル酸はレソルシノールー塩酸法（生化学実験講
座３．脂質の化学１日本生化学会編、 １９７４年＊
ｐ、８８ ）により定量した。〕、ウシ血清を１００と
した場合のシアル酸の回収率は、４２．９％であった。
この両分の主要な蛋白質はウシ血清アルブミンであるが
、ＨＤ抗原糖蛋白質も含まれている。
このウシ血清１／２〜３／４飽和硫安画分をＨＤ抗原糖
蛋白質の代表とする。
（２）ニワトリＨＤ抗体の調製
参考例（２）と同様
（３）ウシ小腸由来アルカリフォスファターゼを用いた
ＨＤ抗原糖蛋白質の測定
エワトＩＪＨＤ抗体を０．０５％ＮａＮ＊ −Ｐ Ｂ
Ｓで、Ａ２１゜＝ｏ、ｏｏｓとなるように希釈し、ポリ
スチレン製９６穴マイクロプレート（イムノプレートＩ
、ヌンク社製）の２７穴に、各式５０μｌずつ加えて４
℃で３日間放置し、エワ）９ＨＤ抗体を各式に結合させ
た。このプレートの各穴中の液を吸引除去した後、０．
０５％Ｔｗｅｅｎ２０−Ｐ’Ｂ Ｓ ３００ ” ４０
０μｌにて各式を３回洗浄し、各穴中の液を充分に除去
し、１％Ｃ０Ａ−０，０５％ＮａＮ５− Ｐ Ｂ Ｓ
３００Ｉ１１を各式に加え、３７℃で２時間放置した。
このプレートの各穴中の液を吸引除去した後、各穴を０
．０５％Ｔｗｅｅｎ２０−Ｐ Ｂ Ｓ ３００〜４００
１１１にて３回洗浄し、各穴中の液を充分除去した。
前記（１）で得たウシ血清１／２〜３／４飽和硫安画分
を１７１０．０００から１／１０，０００ Ｘ ２−＠
まで１％ＣＯＡ −０，０５％ＮａＮ５− Ｐ Ｂ Ｓ
にて倍々希釈し、２穴ずつ計２０穴に５０μｌずつ加え
た。残りの７穴には、１％Ｃ０Ａ−０，０５％ＮａＮ３
− Ｐ Ｂ Ｓ ５０１１１ずっを加えて３７℃で２時
間反応した。このプレートの各穴中の液を吸引除去した
後、０．０５％Ｔｗｅｅｎ ２０−ＰＢＳ３００〜４０
０μｌで７回洗浄し各穴中の液を充分除去し、ウシ小腸
白米アルカＩ７７オス７７ターゼ（Ｐｈｏｓｐａｔａｓ
ｅ ａｌｋａｌｉｎｅ ｆｒｏｍ ｃａｌｆ １ｎｔｅ
ｓｔｉｎｅ ｆｏｒ ｅｎｚｙｍｅｉｍｍｕｎｏａｓｓ
ａｙ （３ｚｙ１０．３ｍ１） ５６７７４４ Ｌｏｔ
、 １０６７５３１−５７７Ｏｃｔ、８７ （ベーリン
〃−・マンハイム・山之内社製）〕を〕１％Ｃ０Ａ−０
．７％ＮａＣ１１ｉ＋Ｍ ＭｙＣｌｔ −３０ｘＭ ）
リエタノールアミン緩衝液（ｐＨ＝７．６）にて１／１
２，５００に希釈した液４０μｌずつを各式に加え３７
℃で３時間反応させた。再び各穴中の液を吸引除去した
後、ｂ＋Ｍ ＭｇＣＴｏ −０，０５％Ｔｗｅｅｎ ２
０− ＰＢＳ３００〜４００μｌで７回洗浄し、各穴中
の液を充分除去して、ＡＬＰローゼ基質液（ジノテスト
社製）を各式に１００μｌずつ加え、３７℃で６０分間
反応させた０次にＡＬＰローゼ呈色液（ジノテスト社製
）を各式に１００μｒずつ加え波長５００ｎｍにおける
吸光度（対照として６３０ｎｗ）をマイクロプレートリ
ーダー（ＭＴＰ−３２（コロナ）〕で測定した。ＨＤ抗
原を添加しない７穴の平均の吸光度を１００とした場合
のウシ血清１／２〜３／４飽和硫安画分を種々の濃度で
加えた各式の吸光度の百分率を算出し、第５図に示した
。
（４）発明の効果
この実験により、ニワ）９ＨＤ抗体に対するウシ小腸由
来アルカリ７オスフアターゼの反応をＨＤ抗原糖蛋白質
が阻害することが明らかとなり、この現象を利用してＨ
Ｄ抗原糖蛋白質を高感度に測定することが可能であるこ
とが明らかとなった。
実施例３
ウシ小腸由来アルカリ７ｔス７アターゼを用いたＨＤ抗
原ガングリオシドの測定（−段階法）（１） ＧＭｓ（
ＮｅｕＧｃ） Ｉｆングリオシドノ調製実施例１−（１
）と同様
このＧＭｓ（ＮｅｕＧｃ） ｆｆングリオシドをＨＤ抗
原ガングリオシドの代表とする。
（２）ニワトーＪＨＤ抗体のａ製
比較例１−（４）と同様
（３）ウシ小腸由来アルカリ７オス７７ターゼを用いた
ＨＤ抗原の測定（二段階法）
前記（２）のニットＩＪ ＨＤ抗体を０．０５％ＮａＮ
３− ＰＢＳで、Ａ２．。＝ｏ、ｏｏｓに希釈し、ポリ
スチレン製９６穴マイクロプレート（イムノプレート１
１ヌンク社製）の３１穴に、各式５０μｌずつ加えて１
０℃で一夜放置し、ニワトリＨＤ抗体を各式に結合させ
た。この各穴中の液を吸引除去し、１％Ｃ０Ａ−０，０
５％ＮａＮ５− Ｐ Ｂ Ｓ ３００１１１を加え、３
７℃で４時間、１０℃で一夜放置した。このプレートの
各式の液を吸引除去した後、０．０５％Ｔｗｅｅｎ２０
− ＰＢＳ３００〜４００μｌにて５回洗浄し、各穴
中の液を充分除去した。 ＧＭ、（ＮｅｕＧｃ） Ｎン
グリオシド１０μｇ／ｘｉから１０ Ｘ ２−’μｇ／
ｗｌまでウシ小腸由来アルカリフォスファターゼ（Ｐｈ
ｏｓｐｂａｔａｓｅ ａｌｋａｌ ｉｎｅｆｒｏｍ ｃ
ａｌｆ １ｎｔｅｓｔｉｎｅ ｆｏｒ ｅｎｚｙａ＋ｅ
ｉｗｕｎｏａｓｓａｙ （３ｍｇ／ ０．３１１ ）
５６７７４４ ＬｏＬ １０ Ｇ ７５３１−５７
１０ｃｔ、 ８７ （ベーリン〃−・□マンハイム・山
之内社製） ） １／２０．０００希釈−１％Ｃ０Ａ−
０，７％ＮａＣｌ−１・ｍＭ ＭｇＣＴｏ −３０ｉＭ
）リエタノールアミン緩衝液（ｐＨ＝７．６）を用い
て、それぞれ倍々希釈し、各々２穴ずつ計１６穴に４０
μｌずつ加え、残りの１５穴には、ＨＤ抗原を含まない
ウシ小腸由来アルカリ７オスフアターゼ（前述）１／２
０，０００希釈−１％Ｃ０Ａ−０，７％ＮａＣｌ −１
ｉ＋ＭＭｇＣ１ｘ −３０ｍＭ ）リエタノールアミン
緩衝液（ｐＨ＝７．６ ） ４０μｌずつ加え、３７℃
で２時間反応さ・せた、各穴中の液を吸引除去し、１ｘ
Ｍ ＭｇＣＴｏ −Ｐ Ｂ ５３００〜４００μｌにで
７回洗浄し、各穴中の液を充分除去した後、１ｍＭ Ｍ
ｇＣＴｏ −Ａ Ｌ Ｐローゼ基質液（ＡＬＰローゼ基
質液（ジノテスト社製）１００ｗｌに対し、１ｘＭ Ｍ
ｇＣＴｏ ０．１０ｗｌを加えで調製した。
〕１００μｌを加え３７℃で６０分間反応させた。
各式にＡＬＰローゼ呈色液（ジノテスト社製）を１０．
０μｌずつ加え、波長５００ｎ−における吸光度（対照
として６３０ｎｍ）をマイクロプレートリーダー（ＭＴ
Ｐ−３２（コロナ）〕で測定した。
ＨＤ抗原を添加しない１５穴の平均の吸光度を１００と
した場合のＧＭａ（ＮｅｕＧｃ） Ｎングリオシドを種
々の濃度で添加した各式の吸光度の百分率を算出し、第
６図に示した。
（４）発明の効果
実施例１では、あらかじめＨＤ抗原ガングリオシドとＨ
Ｄ抗体を反応させた後、ウシ小腸由来アルカリ７オスフ
アターゼを反応させる方法（二段階法）の例を示したが
、本実験ではＨＤ抗原ガングリオシドとウシ小腸由来ア
ルカリ７オスフアターゼを同時にＨＤ抗体と反応させて
も（−段階法）同様に測定し得ることを示した。
実施例４
ウシ小腸由来アルカリ７オスフアターゼを用いたＨＤ抗
／Ｉ；（１１蛋白質の測定（−段階法）（１）ウシ血清
）（Ｄ抗原糖蛋白質画分の調製比較例１−（２）と同様
このウシ血清ＨＤ抗原糖蛋白質画分をＨＤ抗原糖蛋白質
の代表とする。
（２）ニワトリＨＤ抗体の調製
比較例１−（４）と同様
（３）ウシ小腸由来アルカリ７オスフアターゼを用いた
ＨＤ抗原糖蛋白質の測定（−段階法）実施例３−（３）
の０Ｍ３（ＮｅｕＧｃ） ”ングリオシドを、ウシ血清
ＨＤ抗原糖蛋白′！１画分に替えて行った。
結果は、第７図に示した。
（４）発明の効果
実施例２では、あらかじめＨＤ抗原糖蛋白質とＨＤ抗体
を反応させた後、ウシ小腸由来アルカリ７オスフアター
ゼを反応させる方法（二段階法）の例を示したが、本実
験ではＨＤ抗原糖蛋白質とウシ小腸由来アルカリ７オス
フアターゼを同時にＨＤ抗体と反応させても（−段階法
）同様に測定し得ることを示した。[Prior art] Currently, enzyme immunoassay (hereinafter referred to as EIA method)
It is becoming increasingly popular because it is highly sensitive and does not require special equipment. There are two main methods for measuring antigens using the EIA method. They are competitive law and non-competitive law. However, both have their advantages and disadvantages. The latter, ie, non-competitive method, is a measurement method using immobilized antibodies and enzyme-labeled antibodies (so-called sandwich EIA method). This sandwich EIA method is effective for antibodies! By appropriately selecting the enzyme to be detected, the substrate for this enzyme, etc., it is possible to obtain sensitivity comparable to, but even greater than, that of radioimmunoassay (RIA). However, in principle, this 'sandwich EIA method cannot be applied unless the antigen molecule (or antigen particle) that is the test substance has two or more binding sites with antibodies.In contrast, in the competitive method, the sandwich EIA method Although the sensitivity cannot be expected, it can be applied even when the antigen molecule (or antigen particle) that is the test substance has only one binding site with the antibody. By the way, in recent years HD antigen (Hang
anutziu-Deucher'AnLigens
) is attracting attention. The HD antigen refers to a complex sugar chain containing an N-glycolylneuraminic acid (NeuGc) residue as an epitope, and exists as a glycoprotein or ganglioside. On the other hand, HD antibodies are antibodies that react with sugar chains containing N-glycolylneuraminic acid and N-glycolylneuraminic acid residues.HD antigens are widely distributed in the animal kingdom. It is a type of heterophilic antigen and has been thought to be absent in humans and chickens. This HD
It has been revealed that the antigen exists in human and chicken M tumor tissues [Hideyoshi Higashi et al., Lipid Biochemistry Research, Vol. 2
6. p. 92-95, 1984) (H. Higashi et al., J. of Bio
el+emistry (Tokyo)w Vol.
95. p., 785-794, 1984), HD
The development of a highly sensitive and easy-to-use method for measuring antigens is desired. As a method for measuring HD antigen, HDD original ganglioside is extracted and partially purified and Wi1! Developed on I chromatography, vl, prime immunostaining method (H, Higashi
eL al,, J, Biochemistry (
Tokyo) * Vol, 95wp, 1517~
1520, 1984), which is an HD antigen.
Although it is effective for the measurement of glycoside, it cannot be applied to the glycoprotein HD antigen, and the preparation is complicated and is not suitable for handling large quantities of specimens.In addition, we attempted to measure serum HD antigen. As an example, a report by Morito et al.
), this method involves diluting the worm sample serum and diluting it to 100
After heating at °C for 30 minutes, centrifuge to obtain supernatant. Add HD anti-antibody-strength serum to the heated centrifuged supernatant of this serum and let it react at room temperature for 2 hours. Next, add this reaction solution to a 966 microplate to which HD antigen has been bound in advance, let it react, and wash it. After that, alkaline phosphatase (anti-human) IgG or alkaline 777ase at anti-human IgM is added to react, and after washing again, alkaline phosphatase substrate solution is added and reacted, and the absorbance is measured. A method for measuring HD antigen in sample serum by measuring the decrease in absorbance when heated centrifuged supernatant of sample serum is added compared to the absorbance when heated centrifuged supernatant of sample serum is not added. It is. In this method, since the sample serum is heat-treated, it is difficult to receive 117 non-specific reactions derived from HD antibodies and other proteins in the sample serum.On the other hand, most proteins in the sample serum are heated. Since it is insolubilized by the treatment, only a part of the internal content of the HD antigen glycoprotein can be measured, and it is practically difficult to continuously obtain a large amount of HDD positive serum. GM* (NeuGc) W glioside (Kinoshita structure)
Two * GM3 (NeuGc) Ganglioside NeuGc
(22-+3Galβ1-+4GIcβ1→cerNe
uGcα: aN-glycolylneuraminic acid Galβ
: β-lactose Glcβ : β-glucose Cer : Ceramide It has already been reported that chicken HD antibodies, which are effective for HDD measurement, can be obtained by immunizing chickens (Y, F
uji et al., Molecular Imm.
unology, Vol, 19s No, Lp,
87-94.1982), rabbit polyclonal antibody (R, A, La1ne et al., J.
Biol, Chew, 1Vol, 249゜p, 4
460-4466, 1974), mouse monoclonal antibody [Yamazaki + I1hiko et al., Lipid Biochemistry Research, Vol.
29°p., 25-28.1987), human monoclonal antibodies (Osamu Usuba et al. 9, article at the 46th Annual Meeting of the Japanese Cancer Society, p. 304, abstract number 1196.1987). Most enzyme-labeled antigens used in conventional enzyme-linked immunosorbent assays are chemically bonded antigens and enzymes. In this method, it is necessary to chemically treat the antigen or enzyme, and there is a risk of damaging the antigen or enzyme.
, Biohem, Vol, 163y p, 470
~475*1987), but it requires advanced technology and special equipment, and at the current state of the art, it is impossible to change sugar chains by genetic manipulation. That is, enzymatically labeled fiHD antigen cannot be obtained by this method.An attempt was made to enzymatically introduce N-glycolylneuraminic acid residues, that is, HD antigens, into glycoproteins (HoHoHiga & Co., Ltd.). J, C0 Paulson*
J, Biol. Cheta, * Vol, 260. p, 8838-8
849.1985), in principle any enzyme can be
Although it is possible to introduce the antigen enzymatically and prepare the enzymatically labeled ff1HD antigen, this is not done in reality.
Enzyme immunoassays that utilize the antigenicity of enzymes, not just HD antigens, as enzyme-labeled antigens have not previously existed. By the way, most of the proteins in animal bodies are glycoproteins, and many of them have sialic # (N-7 cetylneuraminic acid and N-glycolylneuraminic acid derivatives) residues. Therefore, the enzymes of white rice from humans and other animals other than chickens contain HD antigens to a greater or lesser extent.
In other words, is it originally an enzyme? "aHDHD antigen has the potential to be obtained. Alkaline 7tS7atase derived from bovine small intestine has a high specific activity per molecule, is relatively stable and inexpensive, and is useful for labeling antibodies or antigens. It is widely used as an enzyme. Conventionally, this alkaline 7-osphatase
It has been assumed that sialic acid (N-7 cetylneuraminic acid and N-glycolylneuraminic acid derivatives) residues are not included [HoN, Fernley, The E.
nzymes Vol, ■ (3rd edition
on)w AcademicPress+ 197L
In other words, no one could have guessed that unmodified alkaline 7tS7atase derived from bovine small intestine has a strong affinity for HD antibodies. [Problems to be Solved by the Invention] The present inventors have conducted various studies to address the problems of the prior art, and have concluded that there is no HD antigen in alkaline 7tS7atase derived from bovine small intestine. Despite the fact that the
By identifying this as an X-specific HD antigen, we established a highly sensitive enzyme immunoassay method for HD antigens, and completed the present invention. [Means for Solving the Problems 1] The present invention is a method for measuring HD antigen in a test solution based on the principle of a competitive method using alkali 177 male 77tase derived from bovine small intestine as an alkaline 7 male 77 tase standard aHD antigen. This is an enzyme immunoassay method. That is, bovine small intestine white rice alkal 1
7 male 7 atase as alkaline phosphatase label ia
It is used as an HD antigen, and takes advantage of the fact that the reaction between the HD antigen and HD antibody in the test solution competitively inhibits the reaction between the labeled HD antigen and HD antibody, and also binds to or does not bind to the HD antibody. This is an enzyme immunoassay method for HD antigen, which is characterized in that the amount of HD antigen in a test solution is measured by measuring the amount of alkaline phosphatase derived from bovine small intestine. To further explain the method of the present invention, a test solution and alkaline phosphatase derived from bovine small intestine are reacted sequentially or simultaneously with an HD antibody immobilized on a solid phase such as a microplate, ball, disk piece, or gel. (H) bound to the phase
Amount of alkaline phosphatase (bound to antibody D) or liquid phase! The amount of alkaline 7j
? The amount of HD antigen in the test solution is determined from the 11 standard curve. In addition, after adding HD antibody to the test solution and causing a reaction, or at the same time adding and reacting bovine small intestine white rice alkaline phosphatase, the HD antibody is collected on a solid phase and the amount of bovine small intestine-derived alkaline phosphatase bound to the solid phase is determined. The amount of alkaline phosphatase remaining in the liquid phase was measured by its enzymatic activity, and from the calibration curve of the standard HD antigen solution, the H
Determine the amount of D antigen. At this time, the HD antibodies can be collected on a solid phase by immobilizing an anti-immunoglobulin antibody against the immunoglobulin of white rice animals, or by collecting the HD antibody on a solid phase.
There are methods such as the avidin-biotin method (the HD antibody is biotinylated and avidin is immobilized on the solid phase, and the HD antibody is collected by avidinization and biotin is bound to the solid phase). Furthermore, after adding HD antibody to the test liquid and reacting, or
At the same time, after reacting with alkaline phosphatase derived from bovine small intestine, the HD antibody is aggregated and centrifuged to separate the aggregate and the supernatant. Then, the amount of bovine small intestine polished rice alkaline phosphatase in the aggregate or supernatant was measured by its enzyme activity,
The amount of HD antigen in the test solution is determined from the calibration curve of the standard HD antigen solution. At this time, the HD antibody can be agglutinated by adding an anti-immunoglobulin antibody against im/globulin of white rice animals, or by the avidin-biotin method (the HD antibody is biotinylated and avidin is used alone). Alternatively, there are methods such as polymerization, binding to other proteins, etc. and agglutination, or converting the HD antibody to 7vidin, adding a substance with multivalent biotin such as biotinylated protein, and agglutination). . The bovine small intestine white rice Alka I77 male 7 atase of the present invention is
It can be extracted and purified from bovine small intestine, or purchased commercially, and further purified if necessary. In addition to alkaline phosphatase derived from bovine small intestine, alkali 7 male 77 tase-labeled HD antigen can be obtained using alkaline 7 male 77 tase derived from various animal organs such as bovine kidney, rat liver, rat small intestine white rice, and pig kidney rice. as H
It is also possible to measure D antigen. The HD antibody of the present invention is an antiserum obtained by immunizing a chicken or other animal (@mammary or S class) with an HD antigen (a glycoprotein or ganglioside containing N-glycolylneuraminic acid), or a human ) HD anti-antibody potent serum can be used by purifying human or mouse monoclonal antibodies as necessary. [Operation] According to the present invention, a highly sensitive and simple enzyme immunoassay method for HD antigens is established without the complicated operation of binding antigen and enzyme. EXAMPLES The present invention will be explained below with reference to Examples, but the present invention is not limited thereto in any way. Reference Example: Measurement of affinity between alkaline 7-osphatase derived from bovine small intestine and HD antibody (1) Preparation of GMs (NeuGc) lf glioside Red blood cells were separated from EDTAD-added horse (thoroughbred) blood 101, and hemolyzed with a 1% aqueous acetic acid solution to obtain horse red blood cells. Obtained a membrane ghost. The total lipid fraction was extracted from this horse red blood cell membrane ghost using a chloroform/methanol mixture, and then precipitated with acetone.
5 column chromatography, Saito-Hakomori method (T
, 5aitos S. Hakoeori* J, of Lipid Res
, *Vol, 1'L p, 25'L 1971), GMs(NeuGc) If glioside (structure below) was purified to a standard 0.72. I got it. This GM3 (NeuGc)ff glioside Neu Ge
62-e 3 Galβ1-+4GIcβ1-ec
erNeuGc7: ff-N-glycolylneuraminic acid Galβ: β-lactose Glcβ: β-glucose Cer: Ceramide (2) Preparation of knit IJHD antibody Approximately 1 x g of GMa(NeuGc)N glioside obtained in (1) above, Chicken serum albumin content [Water serum 50% saturated ammonium sulfate supernatant 75% saturated ammonium sulfate precipitated fraction] approx.
0.5ml of purified water and 0.5ml of India Complete Adjuvant (manufactured by Day 7 Co.) was added to emulsify the mixture, and the mixture was injected into the abdomen of chickens (broilers, 30 days old) for immunization. Thereafter, the animals were immunized five times every two weeks in the same manner as described above, and blood was collected at 14 weeks. The collected blood was kept warm at 37°C for 1 hour to coagulate, and then
Antiserum was obtained by centrifugation. Add 172 times the amount of saturated ammonium sulfate solution to this antiserum to salt out and dissolve the antibody components, and after dialysis,
Purified by DEAE-cellulose column chromatography. The chicken rga fraction obtained in this way was
GMs (NeuGc) Octylcef 70-su 4B column (Y, Hirab
ayashi et al., J.Biochem
istry (Tokyo) Vol, 94. p, 32
7 = 330.1983) to obtain a chicken anti-GM3 (NeuGc) antibody (ie, chicken HD antibody). (3) Alkaline phosphatase from bovine small intestine and chicken)
'Measurement of affinity with JHD antibody The chicken HD antibody obtained in (2) above was
=-PBS (sodium chloride 8.0g, potassium chloride 0.20g, phosphoric acid = disodium hydrogen decahydrate 2,
88. , dihydrogen phosphate-potassium 0.2 o, was dissolved in purified water to obtain 1.01. Hereafter referred to as PBS.
), A6. . = 0.010, and polystyrene g196-hole microplate) S (Sumitomo Bake 2
As a control, 0°05% NaN3-PBS containing no antibody was added to 24 wells of Ito Co., Ltd. 1), and each formula 5Q was added to another 24 wells.
ttl was added and left at room temperature overnight to bind the antibody to each well of the plate (no antibody was bound to the wells used as a control). After removing the liquid in each well of this plate by suction, 0.0
Wash each formula three times with 300-400 μl of 5% Tween20-PBS, thoroughly remove the liquid in each well, and add 1% COA.
(Chicken egg albumin, 5 times crystallized (manufactured by Seikagaku Corporation), hereinafter referred to as COA) -0°05% NaN5-
PBS 300111 was added to each formula and left at 37°C for 3 hours. After suctioning and removing the liquid in each hole of this plate, 0.05% Tween20-P B S 300-4
Each formula was washed three times with 00 μl to sufficiently remove the liquid in each well. Bovine small intestine-derived alkaline 7 male 7Tase (Phosphatasealkalkal) was added to each of 4 wells, 2 wells each containing the IJHD antibody and 2 wells each containing the control well.
ine from calf 1ntesti
ne for enzyme iwunoass
ay(3xg10,3x1)567744 Lot, 1
067531-5710ct, 87 (Behrin-Mannheim-Yamanouchisha!I!ri) from 17400 to 1/4
00 x 2-” up to 1% COA-0,7% NaCl-
1zMMgCi'z -30xM) 40μ of a solution diluted with reethanolamine am solution (psummer] = 7.6)
1 of each was added and the mixture was reacted at 37°C for 2 hours. Again, remove the liquid in each hole by suction and add 0.05% Tween20-P.
After washing each formula 7 times with 300 to 400 μl of BS and thoroughly removing the liquid in each well, 100 μl of ALProse substrate solution (manufactured by Ginotest) was added to each formula and incubated at 37°C for 60 minutes.
After reacting for 0 minutes, add 100 μl of ALP rose coloring solution (Ginotest Co., Ltd.) to each formula and wave fl 500.
The absorbance at nm (630 + m as a control) was measured using a microplate reader (MTP-32 (Corona)).
It was measured with The results are shown in FIG. This experiment revealed that Alka-177 osphatase derived from bovine small intestine has strong affinity with chicken HD antibody. Comparative Example I HD anti-antigen Comparison of chemically bound peroxidase and alkaline phosphatase derived from bovine small intestine as a diagnostic HD antigen in HD anti-antigen singlioside measurement (1) GMz (NeuGc) Preparation of W glioside Same as Reference Example (1), this GMs (NeuGc) W glioside is representative of HDD original gangliosides. (2) Preparation of bovine serum HD antigen glycoprotein fraction Bovine serum 1
Add 100 zl of saturated ammonium sulfate to 00111, stir at room temperature for 30 minutes, and centrifuge at 7000 rpm for 30 minutes [RA-7 (
Kubota)], the precipitate (bovine serum globulin) was removed to obtain a supernatant of 162i1. To this supernatant, add 162yl
of saturated ammonium sulfate, stirred at room temperature for 30 minutes, and heated at 7000 rpm.
After removing the supernatant and dissolving the precipitate with purified water, centrifugation (RA-7 (Kubota)) for 2 hours, 0.05% NaN = -
Thoroughly dialyzed against PBS and diluted with 1/2 to 3/3 of bovine serum.
A 4-saturated ammonium sulfate fraction 69.0i+Z was obtained. This bovine serum 1/2 to 3/4 saturated ammonium sulfate fraction (approximately 6511) was added to 50 JIM) Lis-HCl buffer (pH = 8.6).
After dialysis against, the FPLC system (
The column used was Q-Cef70-Scolumn (16X 111alL1), which had strong HDD activity.
using 50zM) lithium hydrochloric acid 11!1t (pH = 8
．． 6) and 100i+M NaCZ-50
i+M) 25 from Lis-HCl buffer fi (pH=8.6)
0zM NaCl-50zM) Lis-HCl buffer (p
H=8.6), flow rate 5mj! /win Grass end elution occurred in 35 minutes. The resulting fractions with strong HDD proactivity were collected and added to 50i+M) lithium salt W1 buffer 1 (pH
= 8.6), dialyzed in the same manner as above, dialyzed against purified water, and lyophilized to obtain bovine serum H.
Approximately 150i+g of D antigen glycoprotein fraction was obtained. The obtained bovine serum HD antigen glycoprotein fraction contains about 7% sialic acid. Sialic acid is produced using the resorcinol-hydrochloric acid method [Biochemistry Experiment Course 3. It was quantified according to Lipid Chemistry 1, edited by the Japanese Biochemical Society, 1974, sp-881. (3) Preparation of HD antigen chemically bound peroxidase Horseradish heroxidase (Grade LLyopl+
1lisat*108090, 3.35zy (manufactured by Mannheim Yamanouchi) to 0.3M NaH
It was dissolved in COs, 1 ml of 1% 2,4-dinitrofluorobenzene suspension vL0 was added, and the mixture was reacted at room temperature for 1 hour. Add 0.5g+1 of 0.06M Nal0+ and react at room temperature for 30 minutes, then add 0.16M ethylene glycol 0.5g+1.
5111 was added and reacted for 1 hour at room temperature, and dialyzed against 10zM sodium carbonate buffer (pH=9.5) at 4°C overnight. To this, 1.631 g of the bovine serum HD antigen glycoprotein fraction obtained in (2) above/101 M carbonate) + 7 um buffer (
Add 0.1 at (pH-9.5) and react at room temperature for 3 hours, then add 0.3 xl of NaBCNH, 1,77u/10zM sodium carbonate buffer 81 (pH = 9.5) and react for an additional 4 hours at room temperature. After that, it was dialyzed against PBS. Add 4 times the amount of purified water to about half of the obtained solution to make 10mM
A DEAE-cellulose column (16 x 83M) equilibrated with potassium hydrogen phosphate buffer (pH = 8.0) was applied, unreacted peroxidase was eluted with 10zM potassium hydrogenphosphate buffer (pH = 8.0), and 20zM ,
301M, 401M, 501M, 60
1M. 70mM potassium hydrogen phosphate buffer (pH=8.0)
Each was sequentially eluted with 1011, and the eluted portion with 30-60i+M potassium hydrogen phosphate buffer (pH = 8.0) was collected and concentrated with Centrif 0-CF-25 (Amicon), and HDD original chemical bonding was performed. Peroxidase. (4) 1!4 preparation of Nit17 HD antibody 0M3 (NeuGc) obtained in (1) above about 1 x g of glioside
. Bovine serum albumin (Cone FrV, manufactured by Armor) 1
xg, 0.5 ml of purified water 1,70 Indo-Humblit Adjuvant (manufactured by Day 7 Co.) 0.5 ij! was added and emulsified, and the mixture was injected into the abdomen of a chicken (broiler, 30 days after breeding) to immunize it. Thereafter, the mice were immunized six times every two weeks in the same manner as described above, and blood was collected at 16 weeks. The collected blood was transferred to Nit 1 in the same manner as in Reference Example (2).
7 HD antibody was obtained. (5) H using HD antigen chemically binding peroxidase
D anti-antigen singlioside measurement Chicken HD antibody obtained in (4) above was added to 0.05% NaN
3-A□ on PBS. Dilute to ~0.010 and place in a polystyrene 96-well microplate (Immunoplate■,
Add 50 μl of each formula to 26 wells of a Nunc (manufactured by Nunc)
The mixture was left overnight at °C to bind the near) IJHD antibody to each formula. The liquid in each hole was removed by suction, and 1% C0A-0,0
After adding 8300 μl of 5% NaN=-P B and leaving it at 37°C for 2 hours, the liquid in each hole was removed by suction, and 0.05
%Tween20-P B S 300-4001tl
After washing 5 times with
0M3(NeuGc) W glioside 10 obtained in 1)
0##/ml, 10Mg7ml1.1μg7ml, 1
00 ng/IIl, 10 ng/xi, 1 ng/m
l, 100 pg 7 ml each 1% C0A-0,05% N
aN=-PBS]! Add 50μl to 2 wells each for a total of 14 wells.
Add 1% C containing no HD antigen to the remaining 12 wells.
0A-0,05% NaN5- P B S 50111
was added and reacted at 37°C for 2 hours. After removing the liquid in each hole by suction, add 0.05% Tween20-P B S 30
The liquid in each hole was sufficiently removed by washing at 0 to 400 ur. In these 26 holes, 1 of HDD original chemically bonded peroxidase is added.
% COA-PBS diluted solution was added to each 40 μl and reacted at 37° C. for 2 hours. After suctioning and removing the liquid in each hole, 0.05
%Tween20-PBS 300~400#j!
Wash 7 times with
I Ivaine buffer (pH = 5.0) 10
Mix 0.33 ml of m1%0.3%Hint and
100 μl of each formula was added and reacted at 37° C. for 60 minutes. Add 50 μr of 4NH230 to these 26 wells, and measure the absorbance at a wavelength of 492 nm (with a wavelength of 630 nm as a control) using Microplate League (MT
P-32 (Corona)]. The average absorbance of the 12 wells that were not reacted with HD antigen was calculated as 1.
The percentage of absorbance of each formula in which GMs(NeuGc):If glioside was reacted at various concentrations was calculated and shown in FIG. (6) Measurement of HDD original ganglioside obtained from bovine small intestine polished rice alkaline phosphatase.
3-PBS, A2. . Dilute to ~0.010 and place in a polystyrene 96-well microplate (Immunoplate 1).
50 μl of each formula was added to 26 wells of a Nunc g1) and left at 10°C overnight to bind the chicken HD antibody to each formula. The liquid in each hole was removed by suction, and 1% C0A-0,
Add 05% NaN5'P B S 300111,
After leaving it at 37°C for 2 hours, the liquid in each hole was removed by suction.
0.05% Tween20-P B S 300 =
After washing 5 times with 4001JI and sufficiently removing the liquid in each hole, the 0M3(NeuGc) If glioside 100/JIF7a 1.10Mg/wl, 1 obtained in (1)
μg7ml, 100ng/ml, 10ng/xi
1%C each of 11 ny/ml and 100 pg/11
Add 50 μl of OA-0,05% NaN3-PBS solution to 2 wells each for a total of 14 wells, and add 1% C0A-0,05% NaN5-P, which does not contain HD antigen, to the remaining 12 wells.
50 μl of B S was added and reacted at 37° C. for 2 hours. After removing the liquid in each hole by suction, add 0.05% Tween20-
PBS 300-4001111 was washed with a trowel to sufficiently remove the liquid in each hole. Into these 26 holes, alkaline 7 male 77 tase derived from bovine small intestine (Phosphatasealka) was added.
l ine from calf 1ntesti
ne for enzyme+ee im+unoa
ssay(3i+g10,3m1)567744 Lo
t, 1067531-5710ct, 87 (Behrin-Mannheim-Yamanouchisha!It)) 1/25
．． 000 dilution 1%C0A-0,7%NaC11i+M
Add 40 μl each of MyClz -30mM) ethanolamine buffer (pH = 7.6) solution 3
The reaction was carried out at 7°C for 2 hours. After removing the liquid in each hole by suction, add 1mM MgC1*-PBS 300-400μ
Wash with 1 wM 7 times to thoroughly remove the liquid in each hole.
MttCTo-ALProse substrate solution (ALProse substrate solution (manufactured by Ginotest) 100 mA'
It was prepared by adding gC1zO, 10ml'. ] was added to each large well and reacted at 37°C for 60 minutes. This 26
Add 100 μl of ALP rose coloring solution (manufactured by Ginotest) to the hole.
The absorbance at a wavelength of 500 nm (630 nm as a control) was measured using a microplate reader [M
TP-32 (Corona)]. The average absorbance of the 12 wells that were not reacted with HD antigen was calculated as 1.
The percentage of absorbance of each formula reacted with various concentrations of GMs (NeuGc) if ganglioside when 00 was calculated and shown in FIG. (7) Results This experiment revealed that when measuring the HD antigen ganglioside, using alkaline 7-os77tase derived from bovine small intestine provides several times higher sensitivity than using peroxidase chemically bound to HD antigen as the enzyme-labeled antigen. Comparative Example 2 Comparison of HD antigen chemically bound peroxidase and bovine small intestine alkaline phosphatase as enzyme labels IHD antigen in HD antigen glycoprotein measurement (1) Bovine blood tffHD
Preparation of Antigen Glycoprotein Fraction As in Comparative Example 1-(2), this bovine serum HD antigen glycoprotein fraction is used as a representative HD antigen glycoprotein. (2) Preparation Comparative Example 1-(3) of HD Antigen Chemically Binding Peroxidase (3) Same as Comparative Example 1-(4) Preparation of Knit 17 HD Antibody (4) HD Using HD Antigen Chemically Binding 5 Peroxidase Measurement of Antigen Glycoprotein Comparative Example 1-(5) The same measurement was carried out except that GM (NeuGc) ganglioside was replaced with bovine serum HD antigen glycoprotein fraction. The results are shown in Figure 3. (5) Measurement of HD antigen glycoprotein using alkaline 7-os 77tase derived from bovine small intestine Comparative example 1 - (6) GM, (NesuGc) :/f Ganglioside bovine serum HD antigen glycoprotein fraction Similar measurements were made. The results are shown in Figure 3. (6) Results This experiment revealed that when measuring HD antigen glycoprotein, using alkaline phosphatase derived from bovine small intestine provides several times higher sensitivity than using peroxyguse chemically bound to HD antigen as an enzyme-labeled antigen. The high usefulness of alkaline phosphatase derived from bovine small intestine has been demonstrated.
Measurement of anti-axy glioside (two-step method) (1) GM
a(NeuGc) if ganglioside preparation reference example (1
), this GMz (NeuGc) If ganglioside is representative of the HD antigen ganglioside. (2) Knit I) HD antibody W4! ! Reference - Submission (2)
Same as (3) Measurement of HD antigen using bovine small intestine white rice Alka 177 male 77tase.
S, A. = o, oos, and place in a polystyrene 96-well microplate (Immunoplate 11
Add 50 μl of each formula to 27 wells of Nunc (manufactured by Nunc) and heat at 4°C.
The cells were left for 3 days to allow chicken HD antibodies to bind to each formula. After suctioning and removing the liquid in each hole of this plate, 0.0
5% Tween20-PBS 300-400μl
Wash each formula 3 times with
%COA ~0.05%NaN5- PBS 30
0 μl was added to each formula and left at 37° C. for 2 hours. After suctioning and removing the liquid in each hole of this plate, each formula is 0.05
%Tween20-P B S 300-400111
The solution in each hole was thoroughly removed by washing three times. Part 20
From 1 μg 7 ml of GMs (NeuGc) If glioside obtained in (1) above to IX 2-' μg 7 ml into the well.
The solution was diluted several times with 1% C0A-0.05% NaN3-PBS, and 5 Ottl was added to each two wells. The remaining 7 holes were filled with 1% C0A-0.05% NaN5-P.
50 μr of B S was added and reacted at 37° C. for 2 hours. After suctioning and removing the liquid in each hole of this plate, 0.0
5% Tween20-PBS 300-400μl
The liquid in each hole was thoroughly removed by washing seven times with
kaline from calf 1ntestin
e for enzyrlleiamunoassay
(3mg70.3ml) 567744 Lot, 106
7531-577Oct, 87 (manufactured by Behrin Mannheim Yamanouchi)] with 11% C0A-07% NaC1
1%M MgCb-30%M) Lieta/-Luaminel
40 μl of a solution diluted to 1/12,500 with Tl buffer (pH=7.6) was added to each formula and reacted at 37° C. for 3 hours. After sucking and removing the liquid in each hole again, add 1% MMg.
cTo −0,05% Tween20−PBS300=
Wash 7 times with 400 μl to thoroughly remove the liquid in each hole.
Add 100% of ALP rose substrate solution (manufactured by Ginotest) to each formula.
0-order ALP was added in microliters and reacted at 37°C for 60 minutes.
Add 100 μl of Rose coloring solution (manufactured by Ginotest) to each formula, and measure the absorbance at 5.500 rm (630 rm as a control) using a microplate reader (MT
P-32 (Corona)]. The percentage absorbance of each formula in which GM and (NeuGc) ganglioside were added at various concentrations was calculated and shown in FIG. 4, assuming that the average absorbance of 7 wells without the addition of HD antigen was 100. (4) Effect of the invention This experiment revealed that HD anti-antigen singlioside inhibits the reaction of bovine small intestinal white rice Alka-177 osphatase against chicken IJHD antibody, and this phenomenon can be used to measure HD anti-rMff glioside with high sensitivity. It became clear that this is possible. Example 2 HD using Alka 177 male 77tase derived from bovine small intestine
Measurement of antigen glycoprotein (two-step method) (1) Bovine serum 172
~374 Preparation of saturated ammonium sulfate fraction in bovine serum 100i1'
Saturated ammonium sulfate 100mm! Add and stir at room temperature for 30 minutes,
After centrifuging at 000 rpm for 30 minutes [RA-7 (Kubota)], the precipitate was removed to obtain 162 ml of supernatant. 162R1 saturated ammonium sulfate was added to this supernatant, stirred at room temperature for 30 minutes, and centrifuged at 7000 rpm for 2 hours. (RA-7 (Kubota))
After dissolving the precipitate with purified water, 0.05% NaN5-
Thoroughly dialyzed against PBS, bovine serum 1/2~3/
A 4-saturated ammonium sulfate fraction of 69.0111 was obtained. 0.05% NaNa of the obtained bovine serum 1/2 to 3/4 saturated ammonium sulfate fraction
-Absorbance at 280 nm in PBS is 34
．． 5, the amount of sialic acid was 1.78 μmole, 7 ml. [Sialic acid is prepared using the resorcinol-hydrochloric acid method (Biochemistry Experiment Course 3. Chemistry of Lipids 1, edited by the Japanese Biochemical Society, 1974*
p, 88). ], the recovery rate of sialic acid was 42.9% when bovine serum was taken as 100. The main protein in both parts is bovine serum albumin, but also contains HD antigen glycoprotein. This bovine serum 1/2 to 3/4 saturated ammonium sulfate fraction is representative of the HD antigen glycoprotein. (2) Preparation of chicken HD antibody Same as reference example (2) (3) Measurement of HD antigen glycoprotein using alkaline phosphatase derived from bovine small intestine Ewat IJ HD antibody was mixed with 0.05% NaN*-P B
Dilute with S so that A21° = o, oos, and place in a polystyrene 96-well microplate (Immunoplate I).
Add 50 μl of each formula to 27 wells of a
The mixture was left at ℃ for 3 days to allow Ewa) 9HD antibody to bind to each formula. After removing the liquid in each hole of this plate by suction, 0.
05% Tween20-P'BS 300" 40
Wash each formula three times with 0 μl, thoroughly remove the liquid in each hole, and add 1% C0A-0,05% NaN5-P B S
300I11 was added to each formula and left at 37°C for 2 hours. After removing the liquid in each hole of this plate by suction, each hole
．． 05% Tween20-P B S 300-400
111 to thoroughly remove the liquid in each hole. The bovine serum 1/2 to 3/4 saturated ammonium sulfate fraction obtained in (1) above was 1710.000 to 1/10,000 x 2-@
Up to 1% COA -0,05% NaN5- P B S
The solution was diluted several times using , and 50 μl of the solution was added to each of 2 wells, for a total of 20 wells. The remaining 7 holes were filled with 1% C0A-0.05% NaN3.
- PBS 50111 was added and reacted at 37°C for 2 hours. After removing the liquid in each hole of this plate by suction, add 0.05% Tween 20-PBS300-40.
Wash 7 times with 0 μl to thoroughly remove the liquid in each hole, and incubate with bovine small intestine white rice Alka I77 male 77tase (Phospatas
e alkaline from calf 1nte
stine for enzyme immunoass
ay (3zy10.3m1) 567744 Lot
, 1067531-577Oct, 87 (manufactured by Behrin Mannheim Yamanouchi)] 1% C0A-0
．． 7%NaC11i+M MyClt-30xM)
1/1 with reethanolamine buffer (pH=7.6)
Add 40 μl of the diluted solution to each formula.
The reaction was carried out at ℃ for 3 hours. After sucking and removing the liquid in each hole again, b+M MgCTo-0,05% Tween 2
Wash 7 times with 300 to 400 μl of 0-PBS, thoroughly remove the liquid in each hole, add 100 μl of ALP rose substrate solution (manufactured by Ginotest) to each formula, and react at 37°C for 60 minutes. ALP rose coloring solution (manufactured by Ginotest Co., Ltd.) was added at 100 μr to each formula and the absorbance at a wavelength of 500 nm (630 nw as a control) was measured using a microplate reader (MTP-32 (Corona)). When the average absorbance is set as 100, the percentage of absorbance of each formula in which bovine serum 1/2 to 3/4 saturated ammonium sulfate fraction was added at various concentrations was calculated and shown in Figure 5. (4) Invention This experiment revealed that the HD antigen glycoprotein inhibits the reaction of bovine small intestine-derived alkaline 7-osphatase to the chicken (chicken) 9HD antibody, and using this phenomenon, H.
It has become clear that it is possible to measure D antigen glycoprotein with high sensitivity. Example 3 Measurement of HD antigen ganglioside using alkaline 7tS7atase derived from bovine small intestine (-step method) (1) GMs (
NeuGc) If glioside preparation example 1-(1
), this GMs (NeuGc) ff glioside is representative of the HD antigen ganglioside. (2) Same as comparative example 1-(4) of chicken JHD antibody (3) Measurement of HD antigen using alkaline 7 male 77 tase derived from bovine small intestine (two-step method) Knit IJ HD antibody of above (2) 0.05% NaN
3- In PBS, A2. . = o, oos and placed in a polystyrene 96-well microplate (Immunoplate 1).
1 Add 50 μl of each formula to 31 wells of Nunc Co., Ltd.).
The chicken HD antibody was allowed to bind to each formula by standing overnight at 0°C. The liquid in each hole was removed by suction, and 1% C0A-0,0
Add 5% NaN5-PBS 300111,
It was left at 7°C for 4 hours and at 10°C overnight. After removing the liquid of each formula from this plate by suction, add 0.05% Tween20.
- Washed 5 times with 300 to 400 μl of PBS to sufficiently remove the liquid in each hole. GM, (NeuGc) N-glioside 10 μg/xi to 10 × 2−′ μg/
Alkaline phosphatase from bovine small intestine (Ph
ospbatase alkal inefrom c
alf 1ntestine for enzya+e
iwunoassay (3mg/0.311)
567744 LoL 10 G 7531-57
10ct, 87 (Berlin〃-・□Manheim-Yamanouchi company)) 1/20.000 dilution-1% C0A-
0.7% NaCl-1・mM MgCTo-30iM
) Using reethanolamine buffer (pH = 7.6), each was diluted two-fold and 40
Add 1/2 microliter of alkaline 7-osphatase (described above) from bovine small intestine, which does not contain HD antigen, to the remaining 15 wells.
0,000 dilution - 1% C0A - 0,7% NaCl -1
i+MMgC1x -30mM) Add 40 μl of reethanolamine buffer (pH=7.6) and incubate at 37°C.
After incubating for 2 hours, the liquid in each hole was removed by suction, and 1x
M
gCTo-ALProse substrate solution (1xM M
It was prepared by adding 0.10 wl of gCTo. ] 100 μl was added and reacted at 37° C. for 60 minutes. 10. Add ALP rose coloring solution (manufactured by Ginotest) to each formula.
Add 0μl each and measure the absorbance at a wavelength of 500n (630nm as a control) using a microplate reader (MT
P-32 (Corona)]. The percentage absorbance of each formula in which GMa(NeuGc)N glioside was added at various concentrations was calculated and shown in FIG. 6, assuming that the average absorbance of 15 wells without the addition of HD antigen was 100. (4) Effect of the invention In Example 1, HD antigen ganglioside and H
An example of a method (two-step method) of reacting with D antibody and then reacting with alkaline 7-osphatase derived from bovine small intestine was shown, but in this experiment, the HD antigen ganglioside and alkaline 7-osphatase derived from bovine small intestine were simultaneously reacted with the HD antibody. It was shown that the measurement can be performed in the same way using the -step method. Example 4 HD anti/I using alkaline 7-osphatase derived from bovine small intestine; (Measurement of 11 proteins (-step method) (1) Bovine serum) (Preparation of D antigen glycoprotein fraction Comparative example 1-(2) and Similarly, this bovine serum HD antigen glycoprotein fraction is used as a representative HD antigen glycoprotein. (2) Preparation of chicken HD antibody Comparative Example 1-Same as (4) (3) HD using alkaline 7-osphatase derived from bovine small intestine Measurement of antigen glycoprotein (-step method) Example 3-(3)
The results were shown in Figure 7. (4) Effects of the Invention In Example 2, the HD antigen We have shown an example of a method (two-step method) in which glycoprotein and HD antibody are reacted and then reacted with alkaline 7-osphatase derived from bovine small intestine. In this experiment, HD antigen glycoprotein and alkaline 7-osphatase derived from bovine small intestine were simultaneously reacted with HD. It was shown that the same measurement can be performed even when reacting with an antibody (-step method).
ＦＡ１図は、参考例の（３）におけるウシ小腸由来アル
カリ７オスフアターゼの希釈率と、その吸光度の関係を
示した。Ｆ−＠は、ニット１ＪＨＤ抗体を結合した穴、
０−０は、抗体の結合のない対照穴でのデータを示した
。
第２図は、比較例１−（５） 、（６）におけるＧＭ、
（ＮｅｕＧｃ ） Ｎングリオシド濃度と、対照穴と比
較した場合の吸光度の百分率との関係を示した。０−−
−０は、ＨＤ抗原化学結合ペルオキシダーゼを標識抗原
として用いた場合を、昏−１は、ウシ小腸由来アルカリ
７オスフアターゼを標識抗原として用−また場合を示し
た。
ｔｊＩＪ３図は、比較例２−（４３、（５）におけるウ
シ血清ＨＤ抗原糖蛋白質画分濃度と、対照穴と比較した
場合の吸光度の百分率との関係を示した。 ０−０は、
ＨＤ抗原化学結合ペルオキシダーゼを標識抗原として用
いた場合を、を−１は、ウシ小腸由来アルカリ７オスフ
アターゼを標識抗原として用νまた場合を示した。
第４図は、実施例１−（３）におけるＧＭｓ（ＮｅｕＧ
ｃ） ｔｌングリオシド濃度と、対照穴と比較した場合
の吸光度の百分率との関係を示した。
第５図は、実施例２−（３）におけるウシ血＊ １／２
〜３７４飽和硫安画分の希釈率と、対照穴と比較した場
合の吸光度の百分率との関係を示した。
第６図は、０Ｍ３（ＮｅｕＧｃ） Ｗ ：／グリオシト
濃度と、対照穴と比較した場合の吸光度の百分率との関
係を示した。
ｔＪ＆７図は、実施例４−（３）におけるウシ血清ＨＤ
抗原糖蛋白質画分濃度と、対照穴と比較した場合の吸光
度の百分率との関係を示した。
特許出願人 株式会社ジノテスト研究所第１図
ウシ小腸由来アルカリ７ｔスフＴターゼ希釈率１／４０
０ Ｘ ２−ｎ希釈
第２図
ＧＭｓ（ＮｅｕＧｃ）ｆｆ ンク’ｊ ｔ シト濃度（
ｎｙ）第３図
ウシ血２ｎＨＤ抗原糖蛋白質画分濃度（ｎｙ）第４図
ＧＭｓ（ＮｅｕＧｃ）ｆｆングリｔシト濃度ｌＸ２−ｎ
μｇｌλ２
ｆＸｒ１５ 図
フシ血清１’７２〜３７４飽和硫安画分希釈率！／４０
．０００ Ｘ ２−ｎ希釈
第６図
０Ｍ３（ＮｅｕＧｃ）ｆｆングリオシド濃度１０Ｘ２−
ｎ μｇ／１１Diagram FA1 shows the relationship between the dilution rate of bovine small intestine-derived alkaline 7-osphatase in reference example (3) and its absorbance. F-@ is a hole bound with Knit 1JHD antibody;
0-0 indicated data from control wells without antibody binding. FIG. 2 shows GM in Comparative Example 1-(5) and (6),
(NeuGc) The relationship between N-glioside concentration and the percentage of absorbance compared to control wells was shown. 0--
-0 indicates the case in which peroxidase chemically bound to HD antigen was used as the labeled antigen, and -1 indicates the case in which alkaline 7-osphatase derived from bovine small intestine was used as the labeled antigen. The tjIJ3 diagram shows the relationship between the concentration of bovine serum HD antigen glycoprotein fraction in Comparative Example 2-(43, (5)) and the percentage of absorbance when compared with the control well. 0-0 is
-1 shows the case when peroxidase chemically bound to HD antigen was used as the labeled antigen, and -1 shows the case when alkaline 7-osphatase derived from bovine small intestine was used as the labeled antigen. FIG. 4 shows GMs (NeuG
c) The relationship between tl nglioside concentration and the percentage of absorbance compared to control wells was shown. Figure 5 shows bovine blood*1/2 in Example 2-(3).
The relationship between the dilution rate of the ~374 saturated ammonium sulfate fraction and the percentage of absorbance compared to the control wells is shown. FIG. 6 shows the relationship between 0M3(NeuGc) W :/gliocyto concentration and percentage of absorbance compared to control wells. tJ&7 figure shows bovine serum HD in Example 4-(3)
The relationship between antigen glycoprotein fraction concentration and percentage of absorbance compared to control wells is shown. Patent Applicant Ginotest Laboratory Co., Ltd. Figure 1 Alkaline 7t Suftase from Bovine Small Intestine Dilution Rate 1/40
0 X 2-n dilution Fig. 2 GMs (NeuGc) ff n'j t Cytoconcentration (
ny) Fig. 3 Bovine blood 2n HD antigen glycoprotein fraction concentration (ny) Fig. 4 GMs (NeuGc) ff Green t cytoconcentration lX2-n
μglλ2 fXr15 Figure Fushi serum 1'72-374 saturated ammonium sulfate fraction dilution rate! /40
．． 000 X 2-n dilution Figure 6 0M3(NeuGc)ff Glioside concentration 10X2-
nμg/11
Claims (1)
スファターゼ標識ＨＤ抗原として用い、被検液中のＨＤ
抗原とＨＤ抗体の反応が、当該標識ＨＤ抗原とＨＤ抗体
との反応を競合的に阻害することを利用し、かつＨＤ抗
体に結合した或いは結合していないアルカリフォスファ
ターゼ量を測定することにより、被検液中のＨＤ抗原量
を測定することを特徴とするＨＤ抗原の酵素免疫測定法
。Using alkaline phosphatase derived from bovine small intestine as an alkaline phosphatase-labeled HD antigen, HD in the test solution was detected.
By utilizing the fact that the reaction between the antigen and HD antibody competitively inhibits the reaction between the labeled HD antigen and HD antibody, and by measuring the amount of alkaline phosphatase bound to or not bound to the HD antibody, the target can be detected. An enzyme immunoassay method for HD antigen, which is characterized by measuring the amount of HD antigen in a test solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4075188A JPH01216267A (en) | 1988-02-25 | 1988-02-25 | Enzyme immunoassay method for hd antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4075188A JPH01216267A (en) | 1988-02-25 | 1988-02-25 | Enzyme immunoassay method for hd antigen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01216267A true JPH01216267A (en) | 1989-08-30 |
Family
ID=12589335
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4075188A Pending JPH01216267A (en) | 1988-02-25 | 1988-02-25 | Enzyme immunoassay method for hd antigen |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01216267A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0539970A2 (en) * | 1991-10-30 | 1993-05-05 | Idemitsu Kosan Company Limited | Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby |
-
1988
- 1988-02-25 JP JP4075188A patent/JPH01216267A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0539970A2 (en) * | 1991-10-30 | 1993-05-05 | Idemitsu Kosan Company Limited | Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby |
| EP0539970A3 (en) * | 1991-10-30 | 1994-05-25 | Idemitsu Kosan Co | Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby |
| US5681729A (en) * | 1991-10-30 | 1997-10-28 | Idemitsu Kosan Company Limited | Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby |
| US5869268A (en) * | 1991-10-30 | 1999-02-09 | Idemitsu Kosan Company Limited | Methods for producing human lymphocytes and human monoclonal antibodies, and human monoclonal antibodies produced thereby |
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