新規間接競合ELISA法によるABO式血液型判定方法の開発とその応用

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Title

新規間接競合ELISA法によるABO式血液型判定方法の開発

_{とその応用( 本文(Fulltext) )}

Author(s)

髙田, 直樹

Report No.(Doctoral

Degree)

博士(農学) 甲第642号

Issue Date

2015-03-13

Type

博士論文

Version

ETD

URL

http://hdl.handle.net/20.500.12099/51016

※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

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ᣂⷙ㑆ធ┹ว

ELISAᴺߦࠃࠆABOᑼⴊᶧဳ್ቯᣇᴺߩ㐿⊒

ߣߘߩᔕ↪

2014ᐕ

ጘ㒂ᄢቇᄢቇ㒮ㅪวㄘቇ⎇ⓥ⑼

↢‛⾗Ḯ⑼ቇ

(ጘ 㒂 ᄢ ቇ)

㜟 ↰ ⋥ ᮸

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1

ᣂⷙ㑆ធ┹ว

ELISAᴺߦࠃࠆABOᑼⴊᶧဳ್ቯᣇᴺߩ㐿⊒

ߣߘߩᔕ↪

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2 ⋡ᰴ ᐨ┨ 1 ABOᑼⴊᶧဳ···5 2 ABOᑼⴊᶧဳߣりర⏕⹺···7 3 ABOᑼⴊᶧဳߣDNAဳ···9 4 ABOᑼⴊᶧဳᬌᩏᴺ ···10 5 ABOᑼⴊᶧဳᬌᩏߩᓸ↢‛ߦࠃࠆ⺋್ቯ···11 6 ᧄ⎇ⓥߩ⋡⊛···12 ╙㧝┨ 㑆ធ┹วELISAᴺߦࠃࠆABOᑼⴊᶧဳᬌᩏᴺߩ㐿⊒ 1-1 ✜⸒ 1-2 㑆ធ┹วELISAᴺߦࠃࠆABOᑼⴊᶧဳᬌᩏᴺߩ㐿⊒ 1-2-1 ታ㛎᧚ᢱ ...14 1-2-2 ↢૕⹜ᢱ(ࡅ࠻ໜᶧ߅ࠃ߮ࡅ࠻ⴊᶧ)ߩ෼㓸 ...15 1-2-3 ⹜ᢱߩ೨ಣℂ...16 1-2-4 㑆ធELISAߦࠃࠆ᛫ේജଔ᷹ቯ...16 1-2-5 㑆ធELISAߦࠃࠆ᛫૕ജଔ᷹ቯ...17 1-2-6 㑆ធ┹วELISAߦࠃࠆໜᶧߩABOᑼⴊᶧဳᬌᩏ...19 1-2-7 㑆ធ┹วELISAߦࠃࠆⴊᶧߩABOᑼⴊᶧဳᬌᩏ...20 1-3 ታ㛎⚿ᨐ 1-3-1 㑆ធELISAߦࠃࠆ᛫ේജଔߩᬌቯ...21 1-3-2 㑆ធELISAߦࠃࠆ᛫૕ജଔߩᬌቯ...22 1-3-3 㑆ធ┹วELISAߦࠃࠆໜᶧߩABOᑼⴊᶧဳᬌᩏ...24

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3 1-3-4 㑆ធ┹วELISAߦࠃࠆⴊᶧߩABOᑼⴊᶧဳᬌᩏ...29 1-4 ⠨ኤ 1-4-1 ࿕⋧ൻ᛫ේ...36 1-4-2 㑆ធELISAߦࠃࠆ࿕⋧ൻ᛫ේജଔ᷹ቯ ...36 1-4-3 㑆ធELISAߦࠃࠆ᛫૕ജଔߩ᷹ቯ...36 1-4-4 㑆ធ┹วELISAߦࠃࠆໜᶧߩABOᑼⴊᶧဳᬌᩏ...37 1-4-5 㑆ធ┹วELISAߦࠃࠆⴊᶧߩABOᑼⴊᶧဳᬌᩏ...37 1-4-6 ⹜ᢱߩ೨ಣℂ...37 1-4-7 ߹ߣ߼ ...38 ╙㧞┨ ABOᑼⴊᶧဳᬌᩏߩ⺋್ቯߦ㑐ਈߔࠆ࿯ფᓸ↢‛ 2-1 ✜⸒...39 2-2 ታ㛎᧚ᢱ෸߮ታ㛎ᣇᴺ ...40 2-2-1 ታ㛎᧚ᢱ ...40 2-2-2 ࿯ფߩណข...40 2-2-3 ⩶ᩣߩಽ㔌...40 2-2-4 D-glycosidaseᵴᕈ᷹ቯ...40 2-2-5 㑆ធ┹วELISAߦࠃࠆBဳ᛫ේಽ⸃ᵴᕈ᷹ቯ ...41 2-2-6 DNA᛽಴ߣ16S rRNAㆮવሶ⸃ᨆ...44 2-3 ታ㛎⚿ᨐ 2-3-1 ࿯ფߩណข...45 2-3-2 ⩶ᩣߩಽ㔌...45 2-3-3 D-glycosidaseᵴᕈ᷹ቯ...45 2-3-4 㑆ធ┹วELISAߦࠃࠆBဳ᛫ේಽ⸃ᵴᕈ᷹ቯ ...48

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4 2-3-5 DNA᛽಴ߣ16S rRNAㆮવሶ⸃ᨆ...50 2-4 ⠨ኤ 2-4-1 ណข⩶ᩣ ...51 2-4-2 D-glycosidaseᵴᕈ...51 2-4-3 㑆ធ┹วELISAߦࠃࠆBဳ᛫ේಽ⸃ᵴᕈ...51 2-4-4 DNA᛽಴ߣ16S rRNAㆮવሶ⸃ᨆ...52 2-4-5 ߹ߣ߼ ...53 ⚳┨ ✚ว⠨ኤ 3-1-1 ↢૕⹜ᢱߩᓸ↢‛ᳪᨴ ...55 3-1-2 ⅣႺᓸ↢‛↱᧪ߩ♧ട᳓ಽ⸃㉂⚛ ...56 3-1-3 ABOᑼⴊᶧဳ᛫ේߩඨቯ㊂⊛ಽᨆᴺ ...56 3-1-4 ࿯ფᓸ↢‛ߦࠃࠆABOᑼⴊᶧဳߩᡷᄌ ...57 3-1-5 ⚿⺰...57 ⻢ㄉ...60 ᒁ↪ᢥ₂...61

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5 ᐨ ᐨ┨ 1 ABOᑼⴊᶧဳ ABOᑼⴊᶧဳߪ, 1900ᐕߦࠝ࡯ࠬ࠻࡝ࠕߩLandsteinerߦࠃࠅႎ๔ߐࠇߡએ᧪, ᦨ߽᦭ ฬߥࡅ࠻ߩㆮવᒻ⾰ߩ㧝ߟߣߒߡ⍮ࠄࠇߡ޿ࠆ20). ABOᑼⴊᶧဳߩ⴫⃻ဳߪ, Table 1ߦ ␜ߒߚㅢࠅߢ, Aဳߩ⿒ⴊ⃿ߦߪAဳ෸߮Hဳ᛫ේ߇ሽ࿷ߒ, ⴊẏߦߪ᛫B᛫૕߇ሽ࿷ߒ ߡ޿ࠆ. ৻ᣇ, Bဳߩ⿒ⴊ⃿ߦߪBဳ෸߮Hဳ᛫ේ߇, ⴊẏߦߪ᛫A᛫૕߇ሽ࿷ߒߡ޿ࠆ. Oဳߩ⿒ⴊ⃿ߦߪHဳ᛫ේ߇, ⴊẏߦߪ᛫A᛫૕߅ࠃ߮᛫B᛫૕߇ሽ࿷ߒߡ߅ࠅ, ABဳߩ ⿒ⴊ⃿ߦߪAဳ, Bဳ෸߮Hဳ᛫ේ߇, ⴊẏߦߪABOᑼⴊᶧဳ᛫ේߦኻߔࠆ᛫૕ߪሽ࿷ߒ ߥ޿.

Table 1 ABO blood group system

ABOᑼⴊᶧဳ᛫ේߪ, Fig. 0-1ߦ␜ߒߚ♧㎮ߢ޽ࠅOဳߩHဳ᛫ේࠍၮᧄ᭴ㅧߣߒ, Hဳ ᛫ේߩᧃ┵ߦ૏⟎ߔࠆFucose(Fuc)ߦ, D -N-acetylgalactosamine (GalNAc)߇D1-3⚿วߒߚ ߽ߩ߇AဳߩAဳ᛫ේ, Galactose (Gal)߇D1-3⚿วߒߚ߽ߩ߇BဳߩBဳ᛫ේߣߥࠆ.

ᣣᧄੱ㓸࿅ߩਛߢߪ, Aဳ㧦Oဳ㧦Bဳ㧦ABဳߩሽ࿷Ყ₸ߪ, ⚂4㧦3㧦2㧦1ߢ޽ࠅ, ઁ

᳃ᣖߣᲧセߒߡ߽஍ࠅ߇ዋߥ޿39)_.

Phenotype Antigen Antibody

A A, H B

B B, H A

O H A, B

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6

Fig. 1 Structure of ABH blood group determinant

ABOᑼⴊᶧဳ᛫ේߪ, ♧⢽⾰޽ࠆ޿ߪ♧࠲ࡦࡄࠢ⾰ߣߒߡ, ⿒ⴊ⃿19)_{ߩߺߥࠄߕ޽ࠄ} ࠁࠆ૕ᶧ, ⚵❱ߦಽᏓߒߡ޿ࠆߎߣ߆ࠄ, ໜᶧ47)_{, ♖ᶧ}3)_{, ⢗ᶧ}21)_{, Ძ㜬}37)_{, 㛽}23)_{, Ὺ}29)_ߥ ߤߩੱ૕ߦ↱᧪ߔࠆ᭽ޘߥㇱ૏ߦಽᏓߒߡ޿ࠆ. ߹ߚ, ࡅ࠻ߪໜᶧ, ♖ᶧߥߤߩಽᴲᶧਛߦ, ABOᑼⴊᶧဳ᛫ේࠍᄙ㊂ߦಽᴲߔࠆಽᴲ ဳߣ, ಽᴲဳߣᲧセߒߡߏߊዋ㊂ߩ᛫ේߩߺಽᴲߔࠆ㕖ಽᴲဳߩ2ㅢࠅߩဳߦಽ㘃ߐࠇ 40)_{, ಽᴲဳߣ㕖ಽᴲဳߪABOᑼⴊᶧဳ᛫ේߩᄙኦߦࠃࠅ᳿ቯߒߡ޿ࠆ. ᣣᧄੱ㓸࿅ߢߪ,} ಽᴲဳ߇⚂8ഀ, 㕖ಽᴲဳ߇⚂2ഀߣߐࠇߡ޿ࠆ. ABOᑼⴊᶧဳߪ, ․ߦක≮㕙ߢ㊀ⷐߥ᛫ේߣߒߡ⹺⼂ߐࠇߡ޿ࠆ. ャⴊߦ↪޿ࠄࠇࠆ ⴊᶧߪ, ㅢᏱ, ABOᑼ෸߮Rhᑼⴊᶧဳ߇หဳߩⴊᶧࠍャⴊߔࠆ18)_{. ߘߩ਄, ⤳ེ⒖ᬀᤨ} ߩ⚵❱ㆡวᬌᩏߦߪ, ABOᑼⴊᶧဳ෸߮HLAဳࠍᬌᩏߒ, ㆡวߩนุࠍ᳿߼ߡ޿ࠆ.

Į-1,2

H

GalNAc

-Į-1,3

A

Gal

-Į-1,3

B

Gal

-Fuc

Gal

-Fuc

Gal

-Fuc

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7 2 ABOᑼⴊᶧဳߣりర⏕⹺ りర⏕⹺ߣߪ, ᄢⷙᮨἴኂ߿‽⟋ߦࠃߞߡ↢ߓߚ, ㆮ૕ߩ⚛ᕈࠍ᣿ࠄ߆ߦߔࠆߎߣࠍ ޿޿, ୘ੱ⼂೎ߣߪ, ޟ↢૕߅ࠃ߮ᱫ૕߹ߚߪߘߩ৻ㇱ߆ࠄ୘ੱࠍ․ቯߔࠆߎߣޠ6)_ࠍ޿ ߁. ߟ߹ࠅ, ୘ੱ⼂೎ࠍⴕ߁ߎߣߦࠃࠅ, りరࠍ᣿ࠄ߆ߦߔࠆߎߣࠍりర⏕⹺ߣ๭ࠎߢ ޿ࠆ. ୘ੱ⼂೎ᴺߦߪ, Ԙචಽߥ୘೎ᕈࠍ߽ߞߡ޿ࠆ(ਇหᕈ. ੱߦࠃߞߡ⇣ߥߞߡ޿ࠆ)ߎ ߣ, ԙ⚳↢ᄌൻ߇ߥ޿(ਇᄌᕈ)ߎߣ, Ԛᬌ⚝ߩߚ߼ߩಽ㘃଻▤߇ଢ೑ߢ޽ࠆߎߣߩ㧟ὐ ߇ᔅⷐߣߐࠇ, ⟑ฬ, ශ┨, ౮⌀, ᜰ⚉ߥߤ߇ฎߊ߆ࠄ೑↪ߐࠇߡ߈ߚ38)_. ୘ੱ⼂೎߇ᔅⷐߣߥࠆ߽ߩߩኻ⽎ߣߒߡ, ↢૕ߢߪ⸥ᙘ༚ᄬ⠪, ᐜఽ, ↥㒮ߢߩขࠅ ㆑߃ߩᣂ↢ఽ߇᜼ߍࠄࠇࠆ. ᱫ૕ߢߪ, ⊕㛽ᱫ૕, ὾ᱫ૕, ಽᢿߐࠇߚᱫ૕ߥߤߢ޽ࠆ 32)_{. ੱ૕ߩ৻ㇱߦ↱᧪ߔࠆ߽ߩߣߒߡߪ, ⴊᶧ, ໜᶧ, ♖ᶧߥߤߩ૕ᶧ߿૕ᶧ᢬, Ძ㜬,} 㛽, ᱤ, ᜰ⚉ߥߤ߇޽ࠆ5, 6)_. ⃻࿷, ᣣᧄ࿖ౝߢ୘ੱ⼂೎ߦ↪޿ࠄࠇߡ޿ࠆᜰᮡߪ, ᜰ⚉, ౮⌀, 㕒⣂ࡄ࠲࡯ࡦ, Ⰲ ᓀ, ✂⤑, ⟑ฬߥߤߩᒻᘒቇ⊛․ᓽߣ, ⴊᶧဳ, DNAߥߤߩㆮવ⊛․ᓽߩ2ߟߦಽ㘃ߢ߈ ࠆ. ᜰ⚉ߪ, ⃻࿷ᦨ߽⏕ታߥ୘ੱ⼂೎ߩᜰᮡߣߥߞߡ޿ࠆ. ࠗࠡ࡝ࠬߩࡋࡦ࡝࡯࡮ࡈࠜ࡯ ࡞࠭ߣ࠙ࠖ࡝ࠕࡓ࡮ࡂ࡯ࠪࠚ࡞ߪ, ᜰߩ㓉✢ߩ⚉᭽߇, ୘ੱ㑆ߢ⇣ߥࠆߎߣ(ਁੱਇห), ᐕ᦬߇⚻ㆊߒߡ߽ᄌൻߒߥ޿(⚳↢ਇᄌ)ߢ޽ࠆߎߣࠍ⑼ቇ⊛ߦ⠨ኤߒ, ⃻࿷ߩᜰ⚉ߩ೑ ↪ߦᄢ߈ߊ⽸₂ߒߚ38)_{. ᜰ⚉ߪ, ฎߊ߆ࠄ‽⟋ᝡᩏ߿りరਇ᣿ᱫ૕ߩりర⏕⹺ߦ೑↪ߐ} ࠇ, ߹ߚ⃻࿷ߢߪ, ㌁ⴕATMߢߩࠠࡖ࠶ࠪࡘࠞ࡯࠼ߩ೑↪, ࠢ࡟ࠫ࠶࠻ࠞ࡯࠼ߩ೑↪, ળ␠߿ࡑࡦ࡚ࠪࡦߥߤߩ౉ㅌ႐, ࡄ࠰ࠦࡦ߿៤Ꮺ㔚⹤ߥߤߩࠦࡦࡇࡘ࡯࠲࡯ࠪࠬ࠹ࡓ ߳ߩࡠࠣࠗࡦߩᧄੱ⏕⹺ߦ߽೑↪ߐࠇߡ޿ࠆ. ᱤ‎ߦࠃࠆ୘ੱ⼂೎ߪ, ᱤߩᴦ≮∥ࠍᲧセߔࠆߎߣߢ୘ੱࠍ․ቯߔࠆᣇᴺߢ޽ࠆ. ᱤ ߪ, ੱ૕ߩਛߢᦨ߽⎬޿⎬⚵❱ߢ޽ࠅ, ᱫᓟ૗ᐕ߽⚻ㆊߒߚᱫ૕ߢ߽, ᱷሽߔࠆน⢻ᕈ

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8 ߇㜞޿. りరਇ᣿ㆮ૕ߩߥ߆ߦߪ, ⋧ᒰߩᐕ᦬ࠍ⚻ㆊߒߚ߽ߩ߽฽߹ࠇࠆߚ߼, ᦭ലߥ ᜰᮡߣߥࠆ. ߹ߚ, ᜰ⚉ߣᱤ‎ߦࠃࠆりర⏕⹺ߪ, ⋥ធ⹜ᢱࠍ⎕უߔࠆߎߣߥߊᬌᩏࠍ ⴕ߃ࠆߎߣ߆ࠄ, 㔡ἴ╬ߩᄙᢙᱫ૕ߩりర⏕⹺ߢᦨ᳢߽↪ߐࠇࠆᣇᴺߪᱤ‎ߣᜰ⚉ߦ ࠃࠆ߽ߩ߇ᄙᢙߢ޽ࠆ. ౮⌀ߦࠃࠆ୘ੱ⼂೎ߦߪ, ฎߊ߆ࠄੱ㑆ߩ⋡ⷞߦࠃࠆ୘ੱߩ․ቯ߇ⴕࠊࠇߡ޿ࠆ. ߹ ߚ, ⃻࿷ߢߪ, ᥧ⸽⇟ภߥߤࠍ೑↪ߔࠆ⍮⼂⹺⸽߿⏛᳇ࠞ࡯࠼ࠍ↪޿ߚ᦭⸽⹺⸽ߦઍࠊ ࠅ, ↢૕⹺⸽, ․ߦ㗻↹௝ߦࠃࠆᧄੱ⏕⹺ᛛⴚ߇᦭ലߦ೑↪ߐࠇߡ޿ࠆ. ↢૕⹺⸽ߩ೑ ὐߪᧄੱߩ․ቯ߇◲ଢߢ޽ࠆߎߣ, ㆙㓒߆ࠄ⹺⸽߇ߢ߈ࠆߎߣ߅ࠃ߮㕖ធ⸅ဳߩ౉ജ ࠺ࡃࠗࠬࠍ↪޿ࠆߎߣ߇ߢ߈ࠆὐߢ޽ࠆ11)_{. ⃻࿷ߢߪ, 㕒⣂⹺⸽, Ⰲᓀ㗻ဳߥߤࠍ↪޿} ߚࡃࠗࠝࡔ࠻࡝ࠢࠬ⹺⸽߇⋓ࠎߦ⎇ⓥߐࠇߡ޿ࠆ45)_{. ߒ߆ߒ, りర⏕⹺ߣ޿߁ὐߢߪ,} ᱫᓟᄌൻ߇ᄢ߈޿ᱫ૕ߥߤߦߪ, ೑↪߇࿎㔍ߢ޽ࠆ. ⴊᶧဳߪ, ೨ㅀߩࠃ߁ߦ, ක≮ߣ‽⟋ᝡᩏߦ߅޿ߡ㊀ⷐߥ᛫ේߢ޽ߞߚ. ⃻࿷ߢߪ, ୘ੱ⼂೎ߩᚻᴺߣߒߡߪᓟㅀߔࠆDNAဳࠍ೑↪ߔࠆߎߣ߇ᄙߊߥࠅ, ᵴべߩᯏળ߇㒢 ࠄࠇߡ߈ߚ. ⴊᶧဳߦߪABOᑼⴊᶧဳࠍߪߓ߼, Lewisᑼ, MNᑼ, Pᑼ, Rhᑼ, ࠳ࡈࠖ࡯ᑼ, ࠠ࠶࠼ᑼߥߤ߇⍮ࠄࠇ, ⇣ห⼂೎ߣ޿߁ὐߢߪ♖ᐲ߇ૐ޿߽ߩߩ, ⷫሶ㐓ቯߥߤߩⴊ✼ 㑐ଥߩಽᨆߦᆭജࠍ⊒ើߔࠆ. ABOᑼⴊᶧဳߪ, ߎࠇࠄߩਛߢ߽, ੱ૕ߦ↱᧪ߔࠆ⚵ ❱࡮૕ᶧ߆ࠄ቟ቯߒߡᬌ಴ߔࠆߎߣ߇ߢ߈ࠆⴊᶧဳߢ޽ࠆ50). DNAဳߦࠃࠆ୘ੱ⼂೎ߪ, 1985ᐕ, ࠗࠡ࡝ࠬߩ࡟ࠬ࠲࡯ᄢቇߩࠕ࡟࠶ࠢ࡮ࠫࠚࡈ࡝࡯ ࠭ߦࠃࠆDNAࡈࠖࡦࠟ࡯ࡊ࡝ࡦ࠹ࠖࡦࠣᴺߩ⊒⴫16)_{ߦࠃࠅ਎⇇⊛ߦᵈ⋡ߐࠇߚ. ᣣᧄ} ߢ߽, ᐔᚑ15ᐕ8᦬ߦߪ, ᣣᧄੱ㓸࿅ߩਛߢᦨ߽಴⃻㗫ᐲ߇㜞޿DNAဳߩ⚵ߺวࠊߖߩ ႐วߦ⚂1100ਁੱߦ1ੱߣ޿߁⏕₸ߢ୘ੱ⼂೎ࠍⴕ߁ߎߣ߇น⢻ߣߥߞߚSTRဳᬌᩏᴺ ߇ዉ౉ߐࠇ60)_{, ᐔᚑ18ᐕ11᦬ߦߪᣂߚߦ6ᐳ૏ࠍㅊടߒ, ᣣᧄੱ㓸࿅ߩਛߢᦨ߽಴⃻㗫} ᐲ߇㜞޿DNAဳߩ⚵ߺวࠊߖߩ႐วߢ⚂4ళ7ජంੱߦ1ੱߣ޿߁⏕₸ߢ୘ੱ⼂೎ࠍⴕ ߁ߎߣ߇น⢻ߣߥߞߡ޿ࠆ61)_.

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9 ୘ੱ⼂೎߇ᔅⷐߣߐࠇࠆ႐ว, ᄙߊߩኻ⽎ߪᱫ૕ߢ޽ࠆߎߣ߇ᄙ޿. ᣣᧄߪ, 1985ᐕ ߩᣣ⥶ࠫࡖࡦࡏᯏ჈⪭੐᡿, 1994ᐕߩਛ⪇⥶ⓨᯏߩฬฎደⓨ᷼჈⪭੐᡿, 1995ᐕߩ㒋␹ ᷆〝ᄢ㔡ἴ, 2004ᐕߩਛ⿧࿾㔡, 2007ᐕߩਛ⿧ᴒ࿾㔡, 2011ᐕߩ᧲ᣣᧄᄢ㔡ἴߩࠃ߁ߦ, ਇᐘߦ߽ᄙᢙߩ‶†⠪ࠍ಴ߔἴኂ࡮੐᡿ࠍ⚻㛎ߒߡ޿ࠆ57). ߎߩࠃ߁ߥ, ᄢἴኂᤨߦ୘ੱ⼂೎ࠍⴕ߁ߚ߼ߦߪ, ߪߓ߼ߦ⹥ᒰ⠪តߒ߆ࠄᆎ߼ࠆᔅ ⷐ߇޽ࠆ. ᄢἴኂᤨߦߪ, ᄙߊߩⴕᣇਇ᣿, ᚲ࿷ਇ᣿⠪ߩ࡝ࠬ࠻߇૞ᚑߐࠇ, ᕈ೎, ⌕ ⴩, ᚲᜬຠ(ㆇォ఺⸵⸽, ࠠࡖ࠶ࠪࡘࠞ࡯࠼, りಽ⸽᣿ᦠ)ߪ, ߎࠇࠄߩ࡝ࠬ࠻߆ࠄ⹥ᒰ ⠪ࠍតߒ಴ߔᄢ߈ߥᚻដ߆ࠅߣߥࠆ. ߒ߆ߒ, ‽⟋ߦࠃࠅனⵝߩน⢻ᕈ߽⠨߃ࠄࠇࠆߎ ߣ߆ࠄ, ή᧦ઙߦᢿቯߔࠆߎߣߪߢ߈ߥ޿. りర⏕⹺ߩ㓙ߦߪ, னⵝߔࠆߎߣ߇ߢ߈ߥ ޿୘ੱߩり૕․ᓽ(ᜰ⚉, ᱤߩᴦ≮∥)߿ㆮવ⊛ᒻ⾰(ⴊᶧဳ߿DNAဳ)ߦࠃࠆ୘ੱ⼂೎ࠍ ⴕ߁ᔅⷐ߇޽ࠆ57)_. ୘ੱ⼂೎ߦߪ, ߹ߕ, ᜰ⚉ߩណข߇ⴕࠊࠇ, ᧄੱߩኻᾖߣߥࠆᜰ⚉ߣߩᲧセ߇ⴕࠊࠇ ࠆ. ὾ᱫ૕߿⊕㛽ᱫ૕ߩࠃ߁ߦᜰ⚉ߩណข߇࿎㔍ߥ႐ว, ᱤ‎ߦࠃࠆᬌᩏ߇ⴕࠊࠇ, ↢ ೨ߩᱤߩᴦ≮⸥㍳ߣㆮ૕ߩᱤߩᴦ≮∥ߣߩᲧセ߇ⴕࠊࠇࠆ. ߎࠇࠄ, り૕․ᓽߦࠃࠆ⏕ ⹺ᣇᴺߪ, ㆮ૕ߣ⹥ᒰ⠪ߩ⸥㍳ߣߩᲧセ࡮ኻᾖߔࠆ⇣ห⼂೎ࠍⴕ߁ᣇᴺߢ޽ࠆ. ᱤߩᴦ ≮∥ߦࠃࠆᾖว߇ߢ߈ߥ޿႐ว߿, ੱ૕ߩ৻ㇱࠍኻ⽎ߣߔࠆ႐วߦߪ, ᓟㅀߔࠆⴊᶧဳ ߿DNAဳᬌᩏࠍⴕ߁. 3 ABOᑼⴊᶧဳߣDNAဳ DNAဳߦࠃࠆ୘ੱ⼂೎ߪ, ⇣ห⼂೎ߩઁ, ⴊ✼㑐ଥߩ⸽᣿ߦ߽ജࠍ⊒ើߔࠆߣ޿߁ ․ᓽ߇޽ࠆ. ߒ߆ߒ, ⴊ✼㑐ଥߦၮߠ޿ߡりర⏕⹺ࠍⴕ߁ߚ߼ߦߪ, りరਇ᣿⠪એᄖߦ, ⴊ✼㑐ଥߦ޽ࠆੱ‛߆ࠄ⹜ᢱࠍណขߔࠆᔅⷐ߇޽ࠆ. ⷫሶ㐓ቯߦߪផቯߐࠇࠆਔⷫ޽ ࠆ޿ߪሶߩ⹜ᢱࠍ, ห⢩㐓ቯߦߪ↵♽ⴊᣖߦ޽ࠆ↵ᕈ, ޽ࠆ޿ߪᲣ♽ⴊᣖⷫߦ޽ߚࠆੱ ‛ߩ⹜ᢱࠍᔅⷐߣߔࠆ. ᄙᢙߩりరਇ᣿ㆮ૕ߣⴊ✼㑐ଥߦ޽ࠆน⢻ᕈߩ޽ࠆੱ‛ోߡ

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10 ߩ⹜ᢱߦߟ޿ߡᬌᩏࠍⴕ߁ߎߣߪ, ല₸ߣᤨ㑆ߩ໧㗴߆ࠄ㕖Ᏹߦ࿎㔍ߢ޽ࠆ. ߹ߚ, ἴ ኂߪ, 㒢ቯߐࠇߚ࿾ၞߦ޿ࠆోߡߩੱޘߦ, ৻ᐲߦⷅ޿ដ߆ࠆ. ߘߩߚ߼, ⴊ✼㑐ଥߦ ޽ࠆኅᣖ߇ోߡⵍἴ⠪ߣߥࠆ႐ว߇ᄙߊ, Ყセ࡮ኻᾖߦᔅⷐߣߥࠆⴊ✼㑐ଥߦ޽ࠆ⠪ߩ ⹜ᢱࠍᓧࠆߎߣ߇࿎㔍ߦߥࠆߎߣ੍߽ᗐߐࠇࠆ. ৻ᣇ, ᣣᧄߦ߅޿ߡߪᐘ޿ߦ߽₂ⴊ೙ᐲ߇᥉෸ߒߡ޿ࠆ. ୘ੱߩABOᑼⴊᶧဳᖱႎ߇ ᴦ≮ᤨ߿₂ⴊᤨߦක≮ᯏ㑐ߦ଻ሽߐࠇߡ޿ࠆน⢻ᕈ߇㜞޿. ା㗬ߢ߈ࠆක≮ᯏ㑐ߩ⸥ ㍳ࠍᓧࠆߎߣߢ߈ࠇ߫, りర⏕⹺ߩኻᾖߣߒߡ೑↪น⢻ߣߥࠅ, ᦭ജߥ୘ੱᖱႎߣߥࠆ. ABOᑼⴊᶧဳߪ, ೨ㅀߩߣ߅ࠅ, ⼂೎♖ᐲߩὐߢߪ, DNAဳߩߘࠇߦߪ෸߫ߥ޿. ߒ߆ ߒ, ABOᑼⴊᶧဳߦࠃࠅ, น⢻ᕈࠍឃ㒰ߔࠆߎߣߪน⢻ߢ޽ࠆߎߣ߆ࠄ, DNAဳ㐓ቯࠍ ⴕ߁೨ߦ, ⹥ᒰ⠪ࠍ⛉ࠅㄟ߻૞ᬺߦㆡߒߡ޿ࠆ. ߹ߚ, ⴊᶧဳ್ቯߦ೑↪ߔࠆⴊᷡቇ⊛ᬌᩏߦᔅⷐߥ⾌↪ߪ, 㕖Ᏹߦ቟ଔߢ޽ࠆߣ޿߁ ೑ὐ߽޽ࠅ, ⃻࿷ߢ߽ABOᑼⴊᶧဳࠍ೑↪ߒߚ୘ੱ⼂೎߿ⷫሶ㐓ቯߪ᦭↪ߥᚻᴺߢ޽ ࠆ. 4 ABOᑼⴊᶧဳᬌᩏᴺ ABOᑼⴊᶧဳᬌᩏߪ, ᓥ᧪߆ࠄಝ㓸⹜㛎, ⸃㔌⹜㛎7, 33, 35, 53)_{, ಝ㓸㒖ᱛ⹜㛎}7)_{, ᷙว⚦}

⢩ಝ㓸ᴺ(mixed cell agglutination reaction: MCAR)59)_{, ㉂⚛఺∉᷹ቯᴺ(Enzyme Linked}

ImmunoSorbent Assay: ELISA)43)_{ߣ޿ߞߚ, ఺∉ቇ⊛ᚻᴺࠍ↪޿ߡABOᑼⴊᶧဳ᛫ේࠍᬌ}

಴ߒߡ޿ߚ. ABOᑼⴊᶧဳㆮવሶ⸃ᨆߦࠃࠆᣇᴺ߽⍮ࠄࠇߡ޿ࠆ߇4, 56)_{, ㆮવሶဳߣ⴫}

⃻ဳ߇ਇ৻⥌ߢ޽ࠆߎߣ߽⠨߃ࠄࠇࠆߎߣ߆ࠄ, ⃻࿷ߢ߽ⴊᷡቇ⊛ᬌᩏߦࠃࠆⴊᶧဳ ್ቯ߇ਥᵹߢ޽ࠆ.

5 ABOᑼⴊᶧဳᬌᩏߩᓸ↢‛ߦࠃࠆ⺋್ቯ

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11 Ⴚߩᓇ㗀ࠍฃߌߡ޿ࠆ. ⊒⷗߇ㆃࠇࠆ߶ߤ, ᬌᩏߦㆡߒߚᬌ૕ߩណข߇࿎㔍ߣߥࠅ, ⴊ ᶧဳ᛫ේߩᬌ಴߇࿎㔍, ޽ࠆ޿ߪ⺋ߞߚဳ߇ᬌ಴ߐࠇࠆߎߣ߇޽ࠆ35)_{. ଀߃߫, ࿯ਛߦ} ၒᴚߒߡ޿ߚⴊ∥߇ⴊᷡቇ⊛ᬌᩏߢߪABဳ, ABOᑼⴊᶧဳㆮવሶ⸃ᨆߢߪBဳߣ್ቯߐ ࠇߚ଀߇᜼ߍࠄࠇࠆ. ߎߩ଀ߢߪ, Acinetobactorዻߩ㧝⩶ᩣߦࠃࠆᳪᨴߦࠃࠅAဳᵴᕈࠍ ₪ᓧߒߚߎߣ߇ႎ๔ߐࠇߡ߅ࠅ, ේ࿃ߣߒߡ⩶૕ᚑಽ↱᧪ߩᄙ♧㘃߿♧࠲ࡦࡄࠢ㘃߇A ဳ᛫ේࠍ᦭ߔࠆߣ⠨߃ࠄࠇߡ޿ࠆ35). ߹ߚ, ৻ㇱߩAspergillusዻߣPenicilliumዻ߇Aဳ᛫ ේࠍ᦭ߒߡ޿ࠆߎߣ߽ႎ๔ߐࠇߡ߅ࠅ63), ߘߩઁߦ߽, ⥄ὼ⇇ߦߪABOᑼⴊᶧဳ᛫ේߣ 㕖Ᏹߦࠃߊૃߚൻቇ᭴ㅧࠍᜬߞߚ⚦⢩ᚑಽࠍ᦭ߔࠆᓸ↢‛߇ሽ࿷ߔࠆߎߣ߇ႎ๔ߐࠇ ߡ޿ࠆ12)_. ෻ኻߦ, ⅣႺਛߩᓸ↢‛↱᧪ߩglycosidaseߦࠃࠅ, ABOᑼⴊᶧဳ᛫ේߩ♧㎮᭴ㅧ߇ಽ ⸃ߐࠇࠆߎߣ߽ႎ๔ߐࠇߡ޿ࠆ. Isekiࠄߦࠃࠇ߫, Aဳ᛫ේߪ, Clostridium tertium14)_{, Bဳ}

᛫ේߪBacillus cereus13)_{, Oဳ᛫ේߪBacillus fulminans}15)_{ߦࠃࠅಽ⸃ߐࠇࠆ. ߐࠄߦ,}

Clostridium perfringens ATCC10543ߩ↥↢ߔࠆendo-E-galactosidaseߦࠃࠅA߅ࠃ߮Bဳ᛫

ේߩਥⷐ㧟♧᭴ㅧ߇ಾᢿߐࠇࠆ଀߽ႎ๔ߐࠇߡ޿ࠆ1). ߽ߒ, ߎࠇࠄߩࠃ߁ߥ㉂⚛ߦࠃ

ࠅ, Aဳ෸߮Bဳ᛫ේߩᧃ┵ߦሽ࿷ߔࠆGalNAc޽ࠆ޿ߪGal߇ಾᢿߐࠇࠆࠃ߁ߥߎߣ߇޽ ࠇ߫, Hဳ᛫ේ߇㔺಴ߔࠆߎߣߦߥࠅ, ⴊᶧဳߪOဳߣ್ቯߐࠇߡߒ߹߁ෂ㒾߇޽ࠆ. ታ 㓙ߦRuminococcus torques strain IX-70ߪ, AဳࠍOဳߦᄌ឵ߔࠆߣႎ๔ߐࠇߡ޿ࠆ22)_.

6 ᧄ⎇ⓥߩ⋡⊛ ⅣႺਛߩᓸ↢‛ߦࠃࠆABOᑼⴊᶧဳ᛫ේߩಽ⸃ࠍේ࿃ߣߔࠆⴊᶧဳᬌᩏߩ⺋್ቯߪ, లಽߦ⿠ߎࠅ߁ࠆ. ߹ߚ, ේ࿃ߣߥࠆᓸ↢‛ߦߟ޿ߡߪ, ޿ߕࠇߩ⎇ⓥ߽ᢿ ⊛ߢ޽ࠅ, ․ߦりరਇ᣿ㆮ૕߇ㆮ⇐ߐࠇࠆߎߣߩᄙ޿࿯ფᓸ↢‛ߦࠃࠆABOᑼⴊᶧဳ᛫ේ߳ߩ૞ ↪ߦߟ޿ߡߪ߶ߣࠎߤᬌ⸛ߐࠇߡ޿ߥ޿. ೨ㅀߒߚࠃ߁ߦ, ᧲ᣣᧄᄢ㔡ἴߩࠃ߁ߥᄢⷙ ᮨἴኂᤨߦABOᑼⴊᶧဳߦࠃࠆ୘ੱ⼂೎߇ᔅⷐߣߥࠆ႐ว߽ᗐቯߐࠇࠆߎߣ߆ࠄ, ⺋

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12 ߞߚ್ቯߩේ࿃ࠍ․ቯߔࠆߎߣ߇ᕆോߢ޽ࠆ. ߘߎߢ, ․ߦ࿯ფ⚦⩶↱᧪ߩglycosidaseߦࠃࠆABOᑼⴊᶧဳ᛫ේߩಽ⸃ߦᵈ⋡ߒߡ, ABOᑼⴊᶧဳ᛫ේߦኻߔࠆ࿯ფᓸ↢‛ߩᓇ㗀ࠍ, ⹦⚦ߦᬌ⸛ߔࠆߎߣߦߒߚ. ߹ߕߪߓ߼ߦ, ABOᑼⴊᶧဳ᛫ේߩಽ⸃ᵴᕈߩ⹏ଔᣇᴺࠍ⏕┙ߔࠆߚ߼ߦ, ᛫ේࠍ․ ⇣⊛߆ߟቯ㊂⊛ߦᬌ಴ߔࠆߎߣ߇ߢ߈ࠆELISAࠍᔕ↪ߒߡ, ABOᑼⴊᶧဳ᛫ේߩඨቯ㊂ ⊛᷹ቯᴺߩ㐿⊒ࠍⴕߞߚ(╙৻┨). ᓥ᧪ߩELISAߦࠃࠆABOᑼⴊᶧဳ್ቯᴺߪ, ⴊ∥ࠍ ⹜ᢱߣߔࠆ႐วߦߪ, ㆡ↪ߢ߈ߥ޿ᰳὐ߇޽ߞߚ߇, ࡊࡠ࠹ࠕ࡯࠯Kࠍ↪޿ߚ࠲ࡦࡄࠢ ಽ⸃෻ᔕࠍ೨ಣℂߣߔࠆߎߣ߅ࠃ߮┹วᴺࠍᔕ↪ߔࠆߎߣߢ, ߶߷ోߡߩ૕ᶧ⹜ᢱ߆ ࠄABOᑼⴊᶧဳ᛫ේߩᬌ಴߇น⢻ߣߥߞߚ. ᧄᴺߦࠃࠅ, ᛫ේߩᷫዋࠍඨቯ㊂⊛ߦ᷹ቯ ߔࠆߎߣ߇น⢻ߣߥࠅ, ࿯ფᓸ↢‛ߦࠃࠆⴊᶧဳᡷᄌᵴᕈࠍ⹏ଔน⢻ߣߒߚ. ᰴߦ, ጘ㒂⋵ਅ58▎ᚲ߆ࠄ࿯ფࠨࡦࡊ࡞ࠍណขߒ, ࡅ࠻ⴊᶧኙᄤၭ࿾ࠍ↪޿ߡᓸ↢‛ 380ᩣࠍන㔌ߒߡ, ታ㓙ߦABOᑼⴊᶧဳ᛫ේ㧔ໜᶧ㧕ߣ෻ᔕߐߖ, ಽ⸃ᵴᕈ᷹ቯࠍⴕߞߚ (╙ੑ┨). Bဳ᛫ේಽ⸃ᵴᕈࠍ␜ߒߚ13⩶ᩣߪ, ోߡBacillusዻߦዻߔࠆ⚦⩶ߢ, ޿ߕࠇ߽ ᬀ‛ߩᩮ࿤߿ᬀ‛૕ߦሽ࿷ߔࠆߎߣ߇᣿ࠄ߆ߦߒߚ. ߎࠇࠄߩౝኈࠍඳ჻⺰ᢥߣߒߡឭ಴ߔࠆޕ

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13 ╙1┨ 㑆ធ┹วELISAᴺߦࠃࠆABOᑼⴊᶧဳᬌᩏᴺߩ㐿⊒ 1-1 ✜⸒ ABOᑼⴊᶧဳߩ್ቯߦߪ, ABOᑼⴊᶧဳ᛫ේࠍᬌ಴ߔࠆᔅⷐ߇޽ࠆ. ᦨ߽◲නߥᚻᴺ ߪ, ⿒ⴊ⃿ߣ᛫ⴊᷡࠍᷙวߒߡಝ㓸෻ᔕࠍⷰኤߔࠆᣇᴺߢ޽ࠆ. ߒ߆ߒ, ṁⴊߒߚⴊᶧ ߿ઁߩ૕ᶧߦ೑↪ߔࠆߎߣ߇ߢ߈ߥ޿. ṁⴊߒߚⴊᶧ߿ઁߩ૕ᶧ߆ࠄ, ABOᑼⴊᶧဳࠍ ᣿ࠄ߆ߦߔࠆߚ߼ߦ, ೨ㅀߩࠃ߁ߦ, ਥߦ⸃㔌⹜㛎, ಝ㓸㒖ᱛ⹜㛎, ᷙวಝ㓸ᴺ╬ߩⴊ ᷡቇ⊛ᬌᩏᴺ߇೑↪ߐࠇߡ޿ࠆ. ߎࠇࠄߩⴊᷡቇ⊛ᬌᩏᴺߦ߅ߌࠆ໧㗴ὐߪ, ⿒ⴊ⃿ߩ ಝ㓸ࠍ⋡ⷞߢⷰኤߔࠆᬌᩏᴺߢ޽ࠆߎߣ߆ࠄ, ቴⷰ⊛ߥ⹏ଔࠍᓧߦߊ޿, ᬌᩏᛛᴺߩ⠌ ᾫߦᤨ㑆߇߆߆ࠆߣ޿ߞߚὐߢ޽ࠆ. ߐࠄߦ, ⃻࿷ߢߪ, ᓥ᧪߆ࠄਥⷐߥ⹜⮎ߣߒߡ ABOᑼⴊᶧဳᬌᩏߦ↪޿ࠄࠇߡ߈ߚࡅ࠻↱᧪ࡐ࡝ࠢࡠ࡯࠽࡞᛫૕߇ੱ㆏਄ߩℂ↱߆ࠄ ౉ᚻ࿎㔍ߣߥࠅ, ࡕࡁࠢࡠ࡯࠽࡞᛫૕ߩ૶↪߇ផᅑߐࠇߡ޿ࠆ. ߘߩ᭽ߥ⁁ᴫߩਛ, ࡕ ࡁࠢࡠ࡯࠽࡞᛫૕ࠍ↪޿ߚELISAߪ, ৻ᐲߦᄙᢙߩᬌ૕ࠍ᷹ቯߔࠆߎߣ߇ߢ߈, ㄦㅦ߆ ߟ᷹ቯኻ⽎‛⾰ࠍ․⇣⊛ߦᬌ಴ߔࠆߎߣ߇น⢻ߢ޽ࠆߎߣ߆ࠄ, ELISAᴺߦࠃࠆⴊᶧဳ ᛫ේᬌ಴ᴺߪ, ABOᑼ9, 17, 28, 31, 44, 54, 55)_{, Lewsᑼ}48, 49)_{, Rhᑼ}27)_{ߩᬌ಴ߦ೑↪ߐࠇߡ޿ࠆ. ᓥ᧪} ߩELISAᴺߦࠃࠆABOᑼⴊᶧဳ᛫ේߩᬌ಴ᴺߪ, ࡑࠗࠢࡠࡊ࡟࡯࠻ߩౝოߦᬌ૕ਛߩ ABOᑼⴊᶧဳ᛫ේࠍ‛ℂ⊛ߦๆ⌕ߐߖߡ࿕⋧ൻߒ, ࡕࡁࠢࡠ࡯࠽࡞᛫૕ߣࡍ࡞ࠝࠠࠪ ࠳࡯࠯ᮡ⼂ߐࠇߚੑᰴ᛫૕ࠍ↪޿ߡᬌ಴ߔࠆ᛫ේ࿕⋧ൻဳߩ㑆ធᴺ߇ਥߦ೑↪ߐࠇߡ ޿ࠆ. ໜᶧ߿♖ᶧ╬ߩ૕ᶧ߇ᬌ૕ߩ႐ว, ABOᑼⴊᶧဳ᛫ේ߇♧࠲ࡦࡄࠢ⾰ߣߒߡሽ࿷ ߔࠆߎߣ߆ࠄ, ࡑࠗࠢࡠࡊ࡟࡯࠻߳ߩ࿕⋧ൻ߇ኈᤃߢ޽ࠆ. ৻ᣇߢ, ⿒ⴊ⃿⤑਄ߩABO ᑼⴊᶧဳ᛫ේߪ♧⢽⾰ߣߒߡሽ࿷ߒߡ޿ࠆߎߣ߆ࠄ8)_{, ㅢᏱߩ✭ⴣᶧߦߪ㔍ṁߢ޽ࠆߎ} ߣ. ߹ߚ, ABOⴊᶧဳ᛫ේࠍ᦭ߔࠆ♧⢽⾰ߣⴊᶧਛߦᄙ㊂ߦሽ࿷ߔࠆࡋࡕࠣࡠࡆࡦ╬ߩ ⴊᶧ↱᧪ߩ࠲ࡦࡄࠢ⾰߇┹ว⊛ߦࡑࠗࠢࡠࡊ࡟࡯࠻ߩო㕙ߦๆ⌕ߐࠇࠆߚ߼, ࿕⋧ൻ ߇లಽߢߪߥ޿ߣߐࠇߡ޿ࠆ41)_{. ߘߩߚ߼, ⴊᶧ⹜ᢱߦߟ޿ߡߪ, ⇇㕙ᵴᕈ೷}2, 30)_߿᦭ᯏ

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14 ṁᇦ62)_{ࠍ೑↪ߒߚ᛽಴ᠲ૞߇ᔅⷐߣߥࠆ߇, ELISA߳ߩᓇ㗀ࠍ⠨ᘦߔࠆߣߎࠇࠄߩ૶↪} ࠍᘕ㊀ߦㆬᛯߔࠆᔅⷐ߇޽ࠆ. ߘߩ⚿ᨐ, ELISAߪ, ໜᶧ߿♖ᶧߣ޿ߞߚ૕ᶧਛߩ࠲ࡦ ࡄࠢᕈߩABOᑼⴊᶧဳ᛫ේߩᬌ಴ߦߩߺߦㆡ↪ߐࠇࠆߎߣߣߥࠅ, ⴊᶧ߿ⴊ∥ਛߩ ABOᑼⴊᶧဳ᛫ේߩᬌ಴ߦߪ, ᓥ᧪ߩⴊᷡቇ⊛ᚻᴺࠍ೑↪ߖߑࠆࠍᓧߥ޿. ߘߩߚ߼, ⹜ᢱߩ⒳㘃ߦᏀฝߐࠇߥ޿ᬌ಴ᚻᴺߩ㐿⊒߇᳞߼ࠄࠇߡ޿ࠆ. ߎߩࠃ߁ߥ໧㗴ࠍ⸃᳿ ߔࠆߚ߼ߦ, ┹วELISAߦᵈ⋡ߒߚ. ┹วELISAߪ, ࿯ფ߿㘩ຠਛߩᱷ⇐ㄘ⮎33, 58)_{, ࠳ࠗ} ࠝࠠࠪࡦ34), ౝಽᴲ߆ߊੂ‛⾰26, 36), ੂ↪⮎‛51)╬ߩ࿕⋧ൻ߇࿎㔍ߢ޽ࠆૐಽሶൻว‛ ߩᬌ಴ߦᔕ↪ߐࠇߡ޿ࠆ. ┹วELISAߪ, ⹜ᢱࠍ⋥ធࡑࠗࠢࡠࡊ࡟࡯࠻ߦ࿕⋧ൻߔࠆᔅ ⷐ߇ߥ޿ߎߣ߆ࠄ, ߎࠇࠍᔕ↪ߔࠆߎߣߦࠃࠅ, ⹜ᢱߩ⒳㘃ߦᏀฝߐࠇߥ޿ABOᑼⴊᶧ ဳ᛫ේߩᬌ಴߇ᦼᓙߢ߈ࠆ. ߘߎߢ, ⹜ᢱߩ⒳㘃ߦࠃࠄߥ޿ABOᑼⴊᶧဳ᛫ේߩᬌ಴ᣇ ᴺߩ⏕┙ࠍ⋡⊛ߣߒߡ, ⹜ᢱߩ೨ಣℂߩᬌ⸛ࠍⴕ޿, 㑆ធ┹วELISAߦࠃࠆABOᑼⴊᶧ ဳ᛫ේߩᬌ಴ᴺߦߟ޿ߡᬌ⸛ࠍⴕߞߚ. 1-2 㑆ធ┹วELISAᴺߦࠃࠆABOᑼⴊᶧဳᬌᩏᴺߩ㐿⊒ 1-2-1 ታ㛎᧚ᢱ

ⴊᶧဳ᛫ේࡊࡠ࡯ࡉ(Blood typeA(tri)-PAA, Blood typeB(tri)-PAA; Glycotech)ߪ, ࡐ࡝ࠕ ࠢ࡝࡞ࠕࡒ࠼ࡐ࡝ࡑ࡯ߦߘࠇߙࠇA, Bဳ᛫ේߩਥⷐ㧟♧᭴ㅧ߇ࠬࡍ࡯ࠨ࡯ࠍ੺ߒߡ⚿ วߒߡ޿ࠆ⵾ຠߢ, Phosphate buffered saline (PBS, pH 7. 5)ߦ1. 0 mg/ mlߦṁ⸃ߒ, ૶↪ᤨ ߦㆡቱPBSߢᏗ㉼ߒߡ↪޿ߚ. Proteinase Kߪ, QIAGEN proteinase K (PK; QIAGEN)ࠍ૶↪ ߒߚ. ࡑࠗࠢࡠࡊ࡟࡯࠻ߪ, F96 MAXISORP NUNC-IMMUNOPLATE (NUNC)ࠍ↪޿, ࡊ ࡟࡯࠻ߩᵞᵺߦߪ0. 05%Tween20ࠍ฽߻PBS (PBS-T)ࠍ↪޿ߚ. ࡉࡠ࠶ࠠࡦࠣṁᶧߪ Bovine serum albumin (BSA; ๺శ⚐⮎Ꮏᬺ)ࠍPBSߦ1. 0%ߦṁ⸃ߒߡ↪޿ߚ(BSA-PBS). ৻ᰴ᛫૕ߪ, ࠟࡦࡑࠢࡠ࡯ࡦ᛫A, B(gamma Biologicals), novaclone Anti A, B(Dominion Biologicals), ࡕࡁࠢࡠ࡯࠽࡞᛫A, Bࡢࠦ࡯(๺శ⚐⮎Ꮏᬺ), ࠮࡜ࠢࡠ࡯ࡦ᛫A, B(Biotest

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15

AG), ࠗ ࡓ ࠦ ࠕ ᛫ A,B ࡕ ࡁ ࠢ ࡠ (IMMUCOR) ෸ ߮ ࡃ ࠗ ࠝ ࠢ ࡠ ࡯ ࡦ ᛫ A, B(Ortho) ࠍ BSA-PBSߢㆡቱᏗ㉼ߒߡ૶↪ߒߚ. ੑᰴ᛫૕ߪ, ࡍ࡞ࠝࠠࠪ࠳࡯࠯ᮡ⼂ࡗࠡ᛫ࡑ࠙ࠬ IgM᛫૕(Peroxidase labeled Goat anti-Mouse IgM, American Qualex)ࠍBSA-PBSߢ1200୚Ꮧ ㉼ߒߡ૶↪ߒߚ. ⊒⦡ၮ⾰ṁᶧߪ, O-ࡈࠚ࠾࡟ࡦࠫࠕࡒࡦ(๺శ⚐⮎Ꮏᬺ)ࠍ0. 5 mg/ml McIlvaine✭ⴣᶧ(pH 5. 0)ߦ⺞ᢛߒ, ෻ᔕ⋥೨ߦ30%ㆊ㉄ൻ᳓⚛᳓ࠍ0. 002%ࠍട߃ߡ૶ ↪ߒߚ.

1-2-2 ↢૕⹜ᢱ(ࡅ࠻ໜᶧ߅ࠃ߮ࡅ࠻ⴊᶧ)ߩ෼㓸

ή૞ὑߦㆬ಴ߒߚABOᑼⴊᶧဳᣢ⍮ߩ38ฬߩஜᐽᚑੱࠃࠅหᗧࠍᓧߡ, ໜᶧ(Aဳಽ ᴲဳ(Se), Aဳ㕖ಽᴲဳ(se)ฦ5ฬ, BဳSeဳ, Bဳseဳฦ5ฬ, OဳSeဳ, Oဳseဳฦ5ฬ, ABဳ Seဳ5ฬ, ABဳseဳ3ฬࠍ෼㓸ߒߚ. ណขᣇᴺߪ, ỹ⚕ ࠍญߦ฽߻ߎߣߦࠃࠅໜᶧࠍᨴ ߺㄟ߹ߖߡឭଏࠍฃߌߚ. ෼㓸ߒߚỹ⚕ ߪ㘑ੇߒ, ቶ᷷ߢ଻ሽߒߚ.

ࡅ࠻ⴊᶧߪ, ૶↪ᦼ㒢ߩಾࠇߚ߽ߩ෸߮㊂ਇ⿷ߦࠃࠅャⴊߦ೑↪ߢ߈ߥ޿ࡅ࠻ⴊᶧ (Aဳ5଀, Bဳ3଀, Oဳ4଀, ABဳ5଀)ࠍ, ᣣᧄ⿒චሼ␠ⴊᶧ࠮ࡦ࠲࡯ࠃࠅ⼑ᷰࠍฃߌ, ታ 㛎㐿ᆎ߹ߢ-80 °Cߩ⿥ૐ᷷ᮏߢ଻ሽߒߚ.

ⴊ∥ߪ5㨪10ᐕߩ㑆, ቶ᷷ߢ଻ሽߒߡ޿ߚߐࠄߒᏓߦઃ⌕ߒߚABOᑼⴊᶧဳᣢ⍮ߩⴊ ∥26଀(Aဳ10଀, Bဳ6଀, Oဳ8଀, ABဳ2଀))ࠍ⹜ᢱߣߒߚ.

ߥ߅, ᧄታ㛎ߩᚻ㗅ߦߟ޿ߡߪ, ጘ㒂ᄢቇ୶ℂክᩏᆔຬળߩᛚ⹺ࠍᓧߚ(ᛚ⹺⇟ภ: 25-167). 1-2-3 ⹜ᢱߩ೨ಣℂ ỹ⚕ ߦណขߒߚໜᶧߪ, ᄢ߈ߐ1×2 cmߦಾᢿߒ, ⚦ಾߒߚᓟ, 250 μlߩPBSߢ᛽಴ࠍ ⴕߞߚ. ⴊᶧߪ, PBSߢ50୚ߦᏗ㉼ߒߚ. ⴊ∥ߪ, ᄢ߈ߐ0. 25×0. 5 cmߦಾᢿߒ, ⚦ಾߒ ߚ ᓟ, 135 μl ߩ PBS ߣ 15 μl ߩ PK ࠍ ᵈ ౉ ߒ ߡ , 56 °C ߢ 2 ᤨ 㑆 Thermomixer compact

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16 (eppendorf)ࠍ↪޿ߡ750 rpmߢᝄߣ߁ߒߡ෻ᔕߐߖṁ಴ߐߖߚ. ߹ߚ, หᄢߩⴊ∥ࠍ 150μlߩPBSߢṁ಴ߒ, PKಣℂࠍታᣉߒߥ޿⹜ᢱࠍኻᾖߣߒߚ. ߥ߅, ᛽಴, ṁ಴, Ꮧ㉼ ᠲ૞ߪኒኽߒߚ1. 5 mlኈߩࡑࠗࠢࡠ࠴ࡘ࡯ࡉౝߢⴕߞߚ. ోߡߩ⹜ᢱ᛽಴ᶧߪ, Thermomixer compactࠍ↪޿ߡ95 °Cߢ15ಽ㑆ᾲಣℂࠍⴕߞߚ. ᾲಣℂᓟ, ㆙ᔃಽ㔌(15000 rpm, 5ಽ㑆)ߒߡ਄ᷡ30 μlࠍ96ⓣࡊ࡟࡯࠻(PCR Plate 96 well PP, Fisher Scientific)ߦ⒖ߒ, ࡊ࡟࡯࠻਄ߢPBSߦࠃࠆᲑ㓏Ꮧ㉼ࠍⴕߞߚ. ಽᴲဳߩໜᶧ ߪ1㨪4096୚, 㕖ಽᴲဳߩໜᶧߪ1㨪64୚, ⴊᶧߪ10㨪3200୚, ⴊ∥ṁ಴ᶧߪ2㨪16୚ߦ Ბ㓏Ꮧ㉼ߒߡ, ታ㛎ߦଏߒߚ. 1-2-4 㑆ធELISAߦࠃࠆ᛫Ḯജଔ᷹ቯ ⴊᶧဳ᛫ේࡊࡠ࡯ࡉࠍછᗧߦᏗ㉼ߒߡ㑆ធELISAࠍⴕ޿, ⴊᶧဳ᛫ේࡊࡠ࡯ࡉߩᦨㆡ Ớᐲࠍᬌ⸛ߒߚ. ࡑࠗࠢࡠࡊ࡟࡯࠻1࠙ࠚ࡞޽ߚࠅ2 μg/ ml߆ࠄ2୚Ბ㓏Ꮧ㉼ߒߚⴊᶧဳ᛫ේࡊࡠ࡯ࡉฦ 50 μlࠍᵈ౉ߒ, 4 °Cߢ㧝᥅࿕⋧ൻߒߚ. ᧂ⚿วߩ᛫ේߪ, ࡑࠗࠢࡠࡊ࡟࡯࠻࠙ࠜ࠶ࠪࡖ ࡯(MW-96CR, BioTec⵾)ࠍ↪޿ߡ, ฦ࠙ࠚ࡞ࠍ250 μl PBS-Tߢ3࿁ᵞᵺߒߡ㒰෰ߒߚ. ࡊ ࡟࡯࠻ࠍᵞᵺᓟ, 1࠙ࠚ࡞޽ߚࠅ100 μlߩBSA-PBSࠍᵈ౉ߒߡ, 37 °Cߢ1 ᤨ㑆ࠗࡦࠠࡘࡌ ࡯࠻ߒߡ, ᛫ේ߇ᧂ⚿วߩო㕙ࠍⷒ޿, ࡉࡠ࠶ࠠࡦࠣࠍⴕߞߚ. ᧂ⚿วߩBSAߪ, ࡊ࡟ ࡯࠻ࠍᵞᵺߒߡ㒰෰ߒ, PBSߢ400୚ߦᏗ㉼ߒߚࡕࡁࠢࡠ࡯࠽࡞᛫૕50 μlࠍᵈ౉ߒ, 37 °Cߢ1 ᤨ㑆෻ᔕߐߖߚ. ᧂ⚿วߩ৻ᰴ᛫૕ߪࡊ࡟࡯࠻ࠍᵞᵺߒߡ㒰෰ߒ, ੑᰴ᛫૕ ߣߒߡࡍ࡞ࠝࠠࠪ࠳࡯࠯ᮡ⼂᛫ࡑ࠙ࠬIgM᛫૕(1200୚Ꮧ㉼, BSA-PBS) 50 μlࠍᵈ౉ߒ, 37 °Cߢ1 ᤨ㑆ࠗࡦࠠࡘࡌ࡯࠻ߒߡ෻ᔕߐߖߚ. ᧂ⚿วߩੑᰴ᛫૕ߪࡊ࡟࡯࠻ࠍᵞᵺߒ ߡ㒰෰ߒ, ⋥ߜߦOPDṁᶧ50 μlࠍᵈ౉ߒ, ㆤశߒߡ, ቶ᷷ߢ10 ಽ㑆෻ᔕߐߖߡ⊒⦡ߒ ߚ. ⊒⦡ᓟ, 1 M ⎫㉄100 μlࠍᵈ౉ߒߡ⊒⦡෻ᔕࠍ஗ᱛߐߖ, ࡊ࡟࡯࠻࡝࡯࠳࡯

(19)

17 ᛫૕ߩઍࠊࠅߦPBSࠍઍ↪ߒߚ߽ߩࠍࡉ࡜ࡦࠢߣߒߚ. 1-2-5 㑆ធELISAߦࠃࠆ᛫૕ജଔ᷹ቯ ⴊᶧဳ᛫ේࡊࡠ࡯ࡉߩỚᐲࠍ1 μg/mlߣߒ, 6⒳ߩࡕࡁࠢࡠ࡯࠽࡞᛫૕ࠍછᗧߦᏗ㉼ߒ ߡ㑆ធELISAࠍⴕ޿, ৻ᰴ᛫૕ߩᦨㆡỚᐲࠍᬌ⸛ߒߚ. ࡑࠗࠢࡠࡊ࡟࡯࠻1࠙ࠚ࡞޽ߚࠅ1μg/mlߩⴊᶧဳ᛫ේࡊࡠ࡯ࡉ50 μlࠍᵈ౉ߒ, 4 °Cߢ 㧝᥅࿕⋧ൻߒߚ. ᵞᵺ, BSA-PBSߦࠃࠆࡉࡠ࠶ࠠࡦࠣߩᎿ⒟ߪ㑆ធELISAߣห᭽ߦⴕߞ ߚ. PBSߢ400ޯ25600୚ߦᏗ㉼ߒߚࡕࡁࠢࡠ࡯࠽࡞᛫૕50 μlࠍᵈ౉ߒ, 37 °Cߢ1 ᤨ㑆෻ ᔕߐߖߚ. ৻ᰴ᛫૕ߩᵞᵺ, ੑᰴ᛫૕ߩᵈ౉, ᵞᵺ, ⊒⦡෻ᔕߪ, 1-2-4ߣห᭽ߦⴕߥߞ ߚ. 1-2-6 㑆ធ┹วELISAߦࠃࠆໜᶧߩABOᑼⴊᶧဳᬌᩏ ໜᶧ᢬ߩṁ಴ᶧࠍ2ಽߒߡ, ৻ᣇߩ30 μlߦ࠮࡜ࠢࡠ࡯ࡦ᛫A᛫૕(800୚Ꮧ㉼, PBS) 30 μlࠍ, ઁᣇߩ30 μlߦࠗࡓࠦࠕ᛫B᛫૕(800୚Ꮧ㉼, PBS) 30 μlࠍട߃ߡ, ߘࠇߙࠇᶧ㊂ࠍ 60 μlߣߒ, 4 °Cߢ1᥅ࠗࡦࠠࡘࡌ࡯࠻ߒߡ, ⹜ᢱਛߩ᛫ේߣ৻ᰴ᛫૕ࠍๆ⌕ߐߖߚ. ࡑࠗࠢࡠࡊ࡟࡯࠻߳ߩⴊᶧဳ᛫ේࡊࡠ࡯ࡉߩ࿕⋧ൻ, ᵞᵺ, BSA-PBSߦࠃࠆࡉࡠ࠶ࠠ ࡦࠣߩᎿ⒟ߪ㑆ធELISAߣห᭽ߦⴕߞߚ. ࿕⋧ൻࠍⴕߞߚࡑࠗࠢࡠࡊ࡟࡯࠻ߦ, ໜᶧ᛽ ಴ᶧߣ৻ᰴ᛫૕ߩᷙวᶧ50 μlࠍᵈ౉ߒ, 37 °Cߢ1 ᤨ㑆෻ᔕߐߖߡ, ৻ᰴ᛫૕ࠍ⹜ᢱਛ ߩ᛫ේߣ࿕⋧ൻ᛫ේߣߩ㑆ߢ┹ว⊛ߦ⚿วߐߖߚ. ᵞᵺߒߡ࠙ࠚ࡞ౝߩ෻ᔕᶧࠍ㒰෰ ᓟ, ࿕⋧ൻ᛫ේߦ⚿วߒߚ৻ᰴ᛫૕ࠍੑᰴ᛫૕ߦࠃߞߡᬌ಴ߒߚ. ߥ߅, ᵞᵺ, ⊒⦡ߪ 㑆ធELISAߣห᭽ߦⴕ޿, ࡊ࡟࡯࠻࡝࡯࠳࡯ߦࠃࠅ490 nmߩๆశᐲࠍ᷹ቯߒߚ. ߥ߅, ৻ᰴ᛫૕ߩઍࠊࠅߦPBSࠍઍ↪ߒߚ߽ߩࠍࡉ࡜ࡦࠢߣߒߚ. ๆశᐲߩB/B0(%)߳ߩᄌ឵ߪᰴᑼߦᓥߞߡⴕߞߚ.

(20)

18

B/B0(%)= (⹜ᢱߩๆశᐲ/⹜ᢱࠍ฽߹ߥ޿߽ߩߩๆశᐲ)100

৻ㅪߩᠲ૞ߪ, Fig. 1-1ߦ␜ߒߚ. ᛫ේᬌ಴ߩ್ቯߪ, B/B0(%)ߩ୯߇90%એਅߦߥࠆ߽

(21)

19

Fig. 1-1Development of an indirect competitive ELISA for the detection of ABO blood group antigens. (a) Samples were diluted with PBS (pH 7. 5), centrifuged at 25,000g for10 min, the supernatant recovered and divided in half. An equal volume (30 μL) of sample was combined with either anti-A or anti-B mAb and incubated at 4 {C for 12 h. (b)The wells of ELISA plates were coated with blood group antigen probes in 50 μL PBS at 4 {C for 12 h. The wells were washed three times with 250 μL PBST. (c) Thewells were blocked with 100 μL BSA–PBS and incubated at 37 {C for 1 h. At the completion of the incubation the wells were washed three times. (d) A 50-μLaliquot containing sample extract combined with either anti-A or anti-B mAb was added to the wells. Plates were incubated at 37 {C for 1 h prior to washing three times. (e) Anti-mouse IgM–HRP conjugate (50 μL) was added to each well. The plates were incubated at 37 {C for 1 h, prior to washing with three times. (f)

o

-Phenylenediamine solution (50 μL) was added to each well, the plates incubated at room temperature for 10 min, and the colorimetric reaction stopped by the addition of100 μL 1 M sulfuric acid was to each well. The OD at 490 nm was measured using a spectrophotometer.

c

d

e _f

b

g

anti-B monoclonal antibody antigen BSA

Anti mouse IgM HRP conjugated

a

(22)

20 1-2-7 㑆ធ┹วELISAߦࠃࠆⴊᶧߩABOᑼⴊᶧဳᬌᩏ ⴊᶧߩᏗ㉼ᶧ෸߮ⴊ∥ߩṁ಴ᶧࠍߘࠇߙࠇ㧞ಽߒߡ, ৻ᣇߩ30 μlߦ࠮࡜ࠢࡠ࡯ࡦ᛫A ᛫૕(800୚Ꮧ㉼, PBS) 30 μlࠍ, ઁᣇߩ30 μlߦࠗࡓࠦࠕ᛫B᛫૕(800୚Ꮧ㉼, PBS) 30 μlࠍ ട߃ߡ, ᶧ㊂ࠍ60 μl ߣߒ, 4 {Cߢ1᥅ࠗࡦࠠࡘࡌ࡯࠻ߒߡ, ⹜ᢱਛߩ᛫ේߣ৻ᰴ᛫૕ࠍ ๆ⌕ߐߖߚ. એਅߩᠲ૞ߪ1㧙2㧙6ߣห᭽ߦⴕߞߚ.

(23)

21 1-3 ታ㛎⚿ᨐ 1-3-1 㑆ធELISAߦࠃࠆ᛫ේജଔߩᬌቯ ⴊᶧဳ᛫ේࡊࡠ࡯ࡉ(Blood typeA(tri)෸߮BloodtypeB(tri))ߩỚᐲࠍ0. 03125 㨪2. 0 μg/ mlߦᄌൻߐߖ, 400୚Ꮧ㉼ߒߚ6⒳(ࠟࡦࡑࠢࡠ࡯ࡦ, ࡁࡧࠔࠢࡠ࡯ࡦ, ࡢࠦ࡯, ࠮࡜ࠢࡠ ࡯ࡦ, ࠗࡓࠦࠕ෸߮ࠝ࡯࠰␠)ߩࡕࡁࠢࡠ࡯࠽࡞᛫A, B᛫૕ࠍ↪޿ߡ㑆ធELISAࠍⴕߞ ߚߣߎࠈ, Aဳࡊࡠ࡯ࡉߦߪ࠮࡜ࠢࡠ࡯ࡦ␠ߩ᛫A᛫૕, Bဳߩࡊࡠ࡯ࡉߦߪࠗࡓࠦࠕ␠ ߩ᛫B᛫૕ࠍ↪޿ߚᤨߦ, ޿ߕࠇ߽1. 0㨪2. 0 μg/ mlߩỚᐲߢๆశᐲ߇ᦨᄢߣߥࠅ, ࡊ࡜ ࠻࡯ߦ㆐ߒߡ޿ߚ(Fig. 1-2, 3). ࠃߞߡ, 1. 0 μg/ mlࠃࠅ㜞Ớᐲߩⴊᶧဳࡊࡠ࡯ࡉࠍ↪޿ࠆ ߎߣߢࡑࠗࠢࡠࡊ࡟࡯࠻ߩ࠙ࠚ࡞ౝო߳ߩ࿕⋧ൻ᛫ේߩ㊂ߪᦨᄢߦߥࠆߣ್ᢿߒߚ.

Fig. 1-2 Detection of blood group A antigens using diluted anti-A six mAbs; Gamma clone (open circle), Novaclone (open square), Wako (open triangle), Seraclone (closed circle), Immucor (closed square) and Ortho (closed triangle),(1:400 dilution). Tests were performed in duplicate. Error bars represent the S. D. highest O. D was observed for Seraclone (closed circle)when the concentration of blood group Type A (tri-PAA) was 1μg/mL.

0.00 1.00 2.00 3.00 0.03125 0.06250 0.12500 0.25000 0.50000 1.00000 2.00000 O .D . 490 n m

(24)

22

Fig. 1-3Detection ofblood group B antigens using diluted anti-B six mAbs. Tests were performed in duplicate. Error bars represent the S. D. The highest O. D was observed for Immucor (closed square) when the concentration of blood group Type B(tri-PAA) was 1 μg/mL

1-3-2 㑆ធELISAߦࠃࠆ᛫૕ജଔߩᬌቯ 6⒳ߩࡕࡁࠢࡠ࡯࠽࡞᛫૕ࠍ400㨪25600୚Ꮧ㉼ߒߚ߽ߩߣ, 1. 0 μg/ ml䈱Ớᐲ䈱ⴊᶧဳ ᛫ේࡊࡠ࡯ࡉࠍ࿕⋧ൻߒߚࡑࠗࠢࡠࡊ࡟࡯࠻ࠍ↪޿ߡ㑆ធELISAᴺࠍታᣉߒߚ. Blood typeA(tri)-PAAࠍ࿕⋧ൻߒߚࡊ࡟࡯࠻ߪ, ޿ߕࠇߩ᛫A᛫૕ࠍ↪޿ߡ߽ᬌ಴߇น⢻ߢ޽ ߞߚ߇, ࠮࡜ࠢࡠ࡯ࡦ␠ߩ᛫A᛫૕ࠍ↪޿ߚᤨ, 400㨪1600୚Ꮧ㉼ߢๆశᐲ߇ᦨᄢߣߥࠅ, ࡊ࡜࠻࡯ߦ㆐ߒߡ޿ߚ(Fig. 1-4). ห᭽ߦ, Blood typeB(tri)-PAAࠍ࿕⋧ൻߒߚࡊ࡟࡯࠻ߪ޿ߕࠇߩ᛫B᛫૕ࠍ↪޿ߡ߽ᬌ ಴߇น⢻ߢ޽ߞߚ߇, ࠗࡓࠦࠕ␠ߩ᛫B᛫૕ࠍ↪޿ߚᤨ, 400㨪1600୚Ꮧ㉼ߢๆశᐲ߇ᦨ ᄢߣߥࠅ, ࡊ࡜࠻࡯ߦ㆐ߒߡ޿ߚ(Fig. 1-5). 0.00 1.00 2.00 3.00 0.03125 0.06250 0.12500 0.25000 0.50000 1.00000 2.00000 O .D . 490 n m

(25)

23

Fig. 1-4Effect of anti-A six mAbs;Gamma clone (open circle), Novaclone (open square), Wako (open triangle), Seraclone (closed circle), Immucor (closed square) and Ortho (closed triangle) concentration on assay sensitivity.Tests were performed in duplicate. Error bars represent the S. D.

Fig. 1-5 Effect of anti-B six mAbs. Tests were performed in duplicate. Error bars represent the S. D. 0.00 1.00 2.00 3.00 1/25600 1/6400 1/1600 1/400 O .D . 490 n m

Monoclonal anti-A antibody dilution

0.00 1.00 2.00 3.00 1/25600 1/6400 1/1600 1/400 O .D . 490 n m

(26)

24 એ਄ߩߎߣ߆ࠄ, ࡑࠗࠢࡠࡊ࡟࡯࠻ߦ࿕⋧ൻߔࠆⴊᶧဳ᛫ේࡊࡠ࡯ࡉߩỚᐲߪ1. 0 μg/ ml, ૶↪䈜䉎 ᛫A᛫૕䈲࠮࡜ࠢࡠ࡯ࡦ␠ߩ᛫A᛫૕ࠍ1600୚Ꮧ㉼, ᛫B᛫૕ߪࠗࡓࠦ ࠕ␠ߩ᛫B᛫૕ࠍ1600୚Ꮧ㉼ߒߚ߽ߩࠍ૶↪ߔࠆߎߣߣߒߚ. 1-3-4 㑆ធ┹วELISAߦࠃࠆໜᶧߩABOᑼⴊᶧဳᬌᩏ ಽᴲဳߩໜᶧ᛽಴ᶧࠍ⹜ᢱߣߒߚ႐วߩ㒖ኂᦛ✢ࠍFig. 1-6ޯ1-9ߦ␜ߔ. B/B0ߩ୯߇ 90%એਅߩ୯ࠍ␜ߒߚฦⴊᶧဳߩᏗ㉼₸ߪ, AဳSeဳໜᶧਛߩAဳ᛫ේ߇1024୚(B/B0=52.

1±28. 9%, Fig. 1-6:Aဳ), BဳSeဳໜᶧਛߩBဳ᛫ේ߇4096୚(B/B0=36. 0±44. 4%, Fig. 1-7:B

ဳ), ABဳSeဳໜᶧਛߩAဳ᛫ේ߇1024୚(A:B/B0=59. 6±17. 8%, Fig. 1-8:Aဳ), ABဳSeဳ

ໜᶧਛߩBဳ᛫ේ߇4096୚(B: B/B0=81. 0±8. 4%, Fig. 1-8:Bဳ)ࠃࠅૐᏗ㉼₸ߢ, ᬌ಴߇น

⢻ߢ޽ߞߚ.

Fig. 1-6 Analysis of type A human secretor (Se) saliva using an indirect

competitiveELISA. Tests were performed in duplicate. Error bars represent the S. D. Human saliva (type A, Se) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle). 0 20 40 60 80 100 120 1/4096 1/1024 1/256 1/64 1/16 1/4 1 B/ B0 (% )

(27)

25

Fig. 1-7Analysis of type B human secretor (Se) saliva. Tests were performed in duplicate. Error bars represent the S. D. Human saliva (typeB, Se) was diluted and combined

with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

Fig. 1-8 Analysis of type AB human secretor (Se) saliva.Tests were performed in duplicate. Error bars represent the S. D. Human saliva (type AB, Se) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

0 20 40 60 80 100 120 1/4096 1/1024 1/256 1/64 1/16 1/4 1 B/ B0 (% )

Dilution ratio of human saliva (blood group AB, secretor) 0 20 40 60 80 100 120 1/4096 1/1024 1/256 1/64 1/16 1/4 1 B/ B0 (% )

(28)

26

Fig. 1-9 Analysis of type O human secretor (Se) saliva. Tests were performed in duplicate. Error bars represent the S. D. Human saliva (typeO, Se) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

㕖ಽᴲဳߩໜᶧ᛽಴ᶧࠍ⹜ᢱߣߒߚ႐วߩ㒖ኂᦛ✢ࠍFig. 1-10ޯ1-13ߦ␜ߔ. B/B0ߩ

୯߇90%એਅߩ୯ࠍ␜ߒߚฦⴊᶧဳߩᏗ㉼₸ߪ, AဳseဳໜᶧਛߩAဳ᛫ේ߇64୚ (B/B0=70. 4±16. 1%, Fig. 1-10:Aဳ), BဳseဳໜᶧਛߩBဳ᛫ේ߇32୚(B/B0=72. 2±13. 5%,

Fig. 1-11:Bဳ), ABဳseဳໜᶧਛߩAဳ᛫ේ෸߮Bဳ᛫ේߩ޿ߕࠇ߽32୚(A:B/B0=74. 4±10. 7%, B: B/B0=55. 3±31. 8%, Fig. 1-12: Aဳ෸߮Bဳ)ࠃࠅૐᏗ㉼₸ߢ, ᬌ಴߇น⢻ߢ޽ߞߚ. 0 20 40 60 80 100 120 1/4096 1/1024 1/256 1/64 1/16 1/4 1 B/ B0 (% )

(29)

27

Fig. 1-10 Analysis of type A human non secretor (se) saliva. Tests were performed in duplicate. Error bars represent the S. D. Human saliva (typeA, se) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

Fig. 1-11 Analysis of type B human non secretor (se) saliva.Tests were performed in duplicate. Error bars represent the S. D. Human saliva (type B, se) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

0 20 40 60 80 100 120 1/64 1/32 1/16 1/8 1/4 1/2 1 B/ B0 (% )

Dilution ratio of human saliva (blood group A, non secretor)

0 20 40 60 80 100 120 1/64 1/32 1/16 1/8 1/4 1/2 1 B/ B0 (% )

(30)

28

Fig. 1-12 Analysis of type AB human non secretor (se) saliva.Tests were performed in duplicate. Error bars represent the S. D. Human saliva (type AB, se) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

Fig. 1-13 Analysis of type O human non secretor (se) saliva. Tests were performed in duplicate. Error bars represent the S. D. Human saliva (type O, se) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

0 20 40 60 80 100 120 1/64 1/32 1/16 1/8 1/4 1/2 1 B/ B0 (% )

Dilution ratio of human saliva (blood group AB, non secretor)

0 20 40 60 80 100 120 1/64 1/32 1/16 1/8 1/4 1/2 1 B/ B0 (% )

(31)

29

1-3-5 㑆ធ┹วELISAߦࠃࠆⴊᶧߩABOᑼⴊᶧဳᬌᩏ

ⴊᶧࠍ⹜ᢱߣߒߚ႐วߩ㒖ኂᦛ✢ࠍFig. 1-14ޯ1-17ߦ␜ߔ. Ꮧ㉼ⴊᶧࠍ⹜ᢱߣߒߚ႐

ว, B/B0ߩ୯߇90%એਅߩ୯ࠍ␜ߒߚฦⴊᶧဳߩᏗ㉼₸ߪ, AဳⴊᶧਛߩAဳ᛫ේ߇800୚

(B/B0=81. 1±7. 0%, Fig. 1-14:Aဳ), BဳⴊᶧਛߩBဳ᛫ේߪ200୚(B/B0=85. 1±3. 6%, Fig.

1-15:Bဳ), ABဳⴊᶧਛߩAဳ᛫ේ߇800୚(B/B0=80. 9±6. 6%, Fig. 1-16:Aဳ), ABဳⴊᶧਛ

ߩBဳ᛫ේߪ200୚(B/B0=68. 6±17. 1%, Fig. 1-16:Bဳ)ࠃࠅૐᏗ㉼₸ߢ޽ࠅ, ᬌ಴߇น⢻ߢ

޽ߞߚ.

Fig. 1-14 Analysis of type A human blood. Tests were performed in duplicate. Error bars represent the S. D. Human Blood (group A) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

0 20 40 60 80 100 120 140 1/3200 1/1600 1/800 1/400 1/200 1/50 1/10 B/ B0 (% )

(32)

30

Fig. 1-15 Analysis of type B human blood. Tests were performed in duplicate. Error bars represent the S. D. Human Blood (group A) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

Fig. 1-16 Analysis of type AB human blood. Tests were performed in duplicate. Error bars represent the S. D. Human Blood (group AB) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

0 20 40 60 80 100 120 140 1/3200 1/1600 1/800 1/400 1/200 1/50 1/10 B/ B0 (% )

Dilution ratio of human blood (type B)

0 20 40 60 80 100 120 140 1/3200 1/1600 1/800 1/400 1/200 1/50 1/10 B/ B0 (% )

(33)

31

Fig. 1-17 Analysis of type O human blood. Tests were performed in duplicate. Error bars represent the S. D. Human Blood (group O) was diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

ⴊ∥ࠍ⹜ᢱߣߒߚ႐วߩ㒖ኂᦛ✢ࠍ, Fig. 1-18ޯ1-21ߦ␜ߔ. B/B0ߩ୯߇90%એਅߩ୯

ࠍ␜ߒߚฦⴊᶧဳߩᏗ㉼₸ߪ, Aဳⴊ∥ਛߩAဳ᛫ේ߇16୚(B/B0=57. 2±16. 5%, Fig.

1-18:Aဳ), Bဳⴊ∥ਛߩBဳ᛫ේ߇16୚(B/B0=55. 6±25. 1%, Fig. 1-19:Bဳ), ABဳⴊᶧਛߩ

Aဳ᛫ේ߇16୚(B/B0=77. 4±1. 7%, Fig. 1-20:Aဳ), ABဳⴊᶧਛߩBဳ᛫ේߪ16୚(B/B0=81.

1±2. 2%, Fig. 1-20:Bဳ)ࠃࠅૐᏗ㉼₸ߢ, ᬌ಴߇น⢻ߢ޽ߞߚ. 0 20 40 60 80 100 120 140 1/3200 1/1600 1/800 1/400 1/200 1/50 1/10 B/ B0 (% )

(34)

32

Fig. 1-18 Analysis of type A human blood stain. Tests were performed in duplicate. Error bars represent the S. D. Human blood stain extracts (typeA) were diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

Fig. 1-19 Analysis of type B human blood stain. Tests were performed in duplicate. Error bars represent the S. D. Human blood stain extracts (typeB) were diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

0 20 40 60 80 100 120 1/16 1/8 1/4 1/2 B/ B0 (% )

Dilution of blood stain extract (blood group A)

0 20 40 60 80 100 120 1/16 1/8 1/4 1/2 B/ B0 (% )

(35)

33

Fig. 1-20 Analysis of type AB human blood stain. Tests were performed in duplicate. Error bars represent the S. D. Human blood stain extracts (typeAB) were diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

Fig. 1-21 Analysis of type O human blood stain. Tests were performed in duplicate. Error bars represent the S. D. Human blood stain extracts (typeO) were diluted and combined with anti-A mAb (closed circle) or anti-B mAb (closed triangle).

0 20 40 60 80 100 120 1/16 1/8 1/4 1/2 B/ B0 (% )

Dilution of blood stain extract (blood group AB)

0 20 40 60 80 100 120 1/16 1/8 1/4 1/2 B/ B0 (% )

(36)

34

ⴊ∥ߦߟ޿ߡ, PKಣℂ෸߮ᾲಣℂࠍታᣉߒߥ޿⹜ᢱࠍ⺞ᢛߒ, ಣℂ⹜ᢱߣߩᲧセࠍⴕ ߞߚ. PK෸߮ᾲಣℂࠍᣉߐߥ߆ߞߚⴊ∥⹜ᢱߢߪ, ⴊ∥ਛߩ᛫ේ߳ߩ৻ᰴ᛫૕ߩ⚿ว߇ ਇචಽߢ޽ߞߚ߇(Fig. 1-22, 1-23), PK෸߮ᾲಣℂߦࠃߞߡ, ⴊ∥ਛߩ᛫ේ߳ߩ৻ᰴ᛫૕ ߩๆ⌕߇⏕⹺ߢ߈ߚ(Fig. 1-22, 1-23: Pk + heat treatment). ߘߩ⚿ᨐ, PK෸߮ᾲಣℂߦࠃߞ ߡⴊ∥߆ࠄᶧਛߦⴊᶧဳ᛫ේ߇චಽߦṁ಴ߐࠇ, ᛫૕ߣߩ⚿ว߇ኈᤃߦߥߞߚߣ⠨߃ ࠄࠇߚ. ߥ߅, PK෸߮ᾲಣℂߪ, Oဳⴊᶧߩᬌᩏߦߪᓇ㗀ࠍ෸߫ߐߥ޿ߎߣ߽⏕⹺಴᧪ ߚ(Fig. 1-24). એ਄ߩ⚿ᨐ߆ࠄ, ᓥ᧪ࠃࠅ࿎㔍ߣߐࠇߡ߈ߚELISAᴺߦࠃࠆⴊ∥ߩABOᑼⴊᶧဳ᛫ ේߩᬌ಴߇น⢻ߦߥࠅ, ↢૕⹜ᢱߦࠃߞߡ᛫ේߩᬌ಴ᣇᴺࠍᄌᦝߔࠆߎߣߥߊAဳ෸߮ Bဳ᛫ේߩᬌ಴߇น⢻ߣߥߞߚ.

Fig. 1-22Effect of PK treatment on the extraction of blood group antigens from type A blood stains. Tests were performed in duplicate. Error bars represent the S. D. Improved

inhibition activity of capture anti-A mAbs was observed for type A blood stains extracted with PK; comparisons made with non PK-treated type A blood stains.

0 20 40 60 80 100 120 1/16 1/8 1/4 1/2 B/ B0

(37)

35

Fig. 1-23 Effect of PK treatment on the extraction of blood group antigens from type B blood stains. Tests were performed in duplicate. Error bars represent the S. D. Improved

inhibition activity of capture anti-B mAbs was observed for type B blood stains extracted with PK; comparisons made with non PK-treated type B blood stains.

Fig. 1-24 Effect of PK treatment on the extraction of blood group antigens from type O blood stains. Tests were performed in duplicate. Error bars represent the S. D.

0 20 40 60 80 100 120 1/16 1/8 1/4 1/2 B/ B0

blood stain dilution(blood group B)

0 20 40 60 80 100 120 1/16 1/8 1/4 1/2 B/ B0

(38)

36 1-4 ⠨ኤ 1-4-1 ࿕⋧ൻ᛫ේ 㑆ធ┹วELISAߩ᷹ቯේℂߪ, ৻ቯ㊂ߩ৻ᰴ᛫૕ߦ, ┹ว᛫ේߣ࿕⋧ൻ᛫ේࠍ┹ว⊛ ߦ⚿วߐߖ, ࿕⋧ൻ᛫ේ஥ߦ⚿วߒߚ৻ᰴ᛫૕ߩ㊂ࠍ᷹ቯߔࠆߎߣߢ┹ว᛫ේߩỚᐲ ࠍ᷹ቯߔࠆ(Fig. 1-1). ߘߩߚ߼, ┹ว᛫ේߣ෻ᔕߒߥ߆ߞߚᧂ෻ᔕߩ৻ᰴ᛫૕ࠍ, น⢻ ߥ㒢ࠅ࿕⋧ൻ᛫ේߦ⚿วߐߖࠆߚ߼ߦߪ, ࿕⋧ൻ᛫ේࠍࡑࠗࠢࡠࡊ࡟࡯࠻ߩ࠙ࠚ࡞ౝ ოߦ㜞ኒᐲߦ⚿วߐߖࠆߎߣ߇᳞߼ࠄࠇࠆ. ᧄᴺߢ࿕⋧ൻ᛫ේߦ೑↪ߒߚᏒ⽼ߩABO ᑼⴊᶧဳ᛫ේࡊࡠ࡯ࡉߪ, ࠕࠢ࡝࡞ࠕࡒ࠼ࡐ࡝ࡑ࡯ߦA, Bဳ᛫ේߩਥⷐ3♧᭴ㅧ߇ࠬࡍ ࡯ࠨ࡯ࠍ੺ߒߡ⚿วߒߚ߽ߩߢ, 㜞ኒᐲߦࡑࠗࠢࡠࡊ࡟࡯࠻߳࿕⋧ൻ߇ኈᤃߦ㆐ᚑߢ ߈ࠆߎߣ߇⏕⹺ߢ߈ߚ. 1-4-2 㑆ធELISAߦࠃࠆ࿕⋧ൻ᛫ේജଔ᷹ቯ ABOᑼⴊᶧဳ᛫ේࡊࡠ࡯ࡉߩỚᐲࠍ0. 03125㨪2 μg/mlߦᄌൻߐߖ, 6⒳㘃ߩࡕࡁࠢࡠ ࡯࠽࡞᛫૕(400୚Ꮧ㉼)ࠍ↪޿ߡ, 㑆ធELISAᴺߦࠃߞߡ࿕⋧ൻߔࠆⴊᶧဳ᛫ේࡊࡠ࡯ ࡉߩᦨㆡỚᐲߦߟ޿ߡᬌ⸛ߒߚ. ABOᑼⴊᶧဳ᛫ේࡊࡠ࡯ࡉߪ, ࠮࡜ࠢࡠ࡯ࡦ␠ߩ᛫A ᛫૕, ࠗࡓࠦࠕ␠ߩ᛫B᛫૕ࠍ↪޿ߚ႐วߦ, ߘࠇߙࠇๆశᐲ߇ᦨᄢߣߥߞߚ1.0 μg/ ml ߩỚᐲߦ⺞ᢛߒߡ, 50μlࠍࡑࠗࠢࡠࡊ࡟࡯࠻ߩ࠙ࠚ࡞ߦᵈ౉ߒߡ࿕⋧ൻࠍⴕ߁ߎߣߣߒ ߚ(Fig. 1-2, 3). 1-4-3 㑆ធELISAߦࠃࠆ᛫૕ജଔߩ᷹ቯ ⴊᶧဳ᛫ේࡊࡠ࡯ࡉߩỚᐲࠍ1. 0 μg/ mlߣߒ, 6⒳ߩࡕࡁࠢࡠ࡯࠽࡞᛫૕ߩᏗ㉼୚₸ࠍ ᄌ ൻ ߐ ߖ, 㑆ធ ELISAᴺ ߦ ࠃࠅ ᦨㆡ ߥ᛫ ૕ ߩᏗ ㉼ ୚₸ ࠍᬌ ⸛ߒ ߚ ߣߎ ࠈ , Blood typeA(tri)-PAAߦߪ1600Ꮧ㉼ߩ࠮࡜ࠢࡠ࡯ࡦ␠ߩ᛫A᛫૕, Blood typeB(tri)-PAAߦߪ1600 ୚Ꮧ㉼ߩࠗࡓࠦࠕ␠ߩ᛫B᛫૕߇ᦨ߽෻ᔕߦㆡߒߡ޿ߚ(Fig. 1-4,1-5).

(39)

37

1-4-4 㑆ធ┹วELISAߦࠃࠆໜᶧߩABOᑼⴊᶧဳᬌᩏ

ໜᶧࠍ⹜ᢱߣߒߚ႐ว, ಽᴲဳߢߪ, Aဳ෸߮ABဳ߇1024୚, Bဳ߇4096୚Ꮧ㉼߹ߢ

(Fig. 1-6, 7, 8), 㕖ಽᴲဳߢߪAဳ߇64୚, Bဳ෸߮ABဳߣ߽ߦ32୚Ꮧ㉼B/B0ߩ୯߇90%એ

ਅߣߥࠅ, 㓁ᕈߣ್ቯߐࠇߚ(Fig. 1-10, 11, 12). ߒߚ߇ߞߡ, ᧄᴺߦࠃࠆໜᶧਛߩABO ᑼⴊᶧဳ᛫ේߩᬌ಴ᗵᐲߪ, ಽᴲဳߢ߅߅ࠃߘ1024୚Ꮧ㉼߹ߢ, 㕖ಽᴲဳߢ߅߅ࠃߘ32 ୚Ꮧ㉼߹ߢߣ⠨߃ࠄࠇߚ. 1-4-5 㑆ធ┹วELISAߦࠃࠆⴊᶧߩABOᑼⴊᶧဳᬌᩏ ṁⴊ╬ߩᓇ㗀ߦࠃࠅ, ⿒ⴊ⃿ࠍᓧࠆߎߣ߇ߢ߈ߥ޿⹜ᢱࠍᗐቯߒ, ಓ⚿ᓟ, ⸃ಓߒߡ ṁⴊߐߖߚࡅ࠻ⴊᶧߦߟ޿ߡ, ᧄᴺࠍ↪޿ߚABOᑼⴊᶧဳ᛫ේߩᬌ಴ࠍⴕߞߚ. ⴊᶧࠍ⹜ᢱߣߒߚ႐ว, Aဳߪ800୚Ꮧ㉼߹ߢ, Bဳߪ200୚Ꮧ㉼, ABဳߪ200୚Ꮧ㉼߹ ߢ㓁ᕈߩ෻ᔕࠍ␜ߒߚ(Fig. 1-14, 15, 16). ߒߚ߇ߞߡ, ᧄᴺߦࠃࠆⴊᶧਛߩABOᑼⴊᶧ ဳ᛫ේߩᬌ಴ᗵᐲߪ, ߅߅ࠃߘ200୚Ꮧ㉼߹ߢߣ⠨߃ࠄࠇߚ. ߥ߅, 10㨪400୚Ꮧ㉼ߩA ဳⴊᶧߦኻߔࠆ᛫B᛫૕, 10㨪400୚Ꮧ㉼ߩBဳⴊᶧߦኻߔࠆ᛫A᛫૕෸߮10㨪400୚Ꮧ㉼ ߩOဳⴊᶧߦኻߔࠆ᛫A, B᛫૕ߩB/B0%ߪ, 100%ࠍᄢ߈ߊ⿥߃ࠆ୯ࠍᬌ಴ߒߚ(Fig. 1-14, 15, 17). ߎࠇߪ, ⴊᶧߩᏗ㉼₸߇ᄢ߈ߊߥࠆ߶ߤ୯߇ዊߐߊߥߞߡ޿ࠆߎߣ߆ࠄ, ᾲಣ ℂߦࠃࠅචಽߦࡋࡕࠣࡠࡆࡦࠍ㒰෰ߒ߈ࠇߥ߆ߞߚߚ߼, 㜞Ớᐲߩࡋࡕࠣࡠࡆࡦ߇ ELISAߦᓇ㗀ࠍਈ߃ߚߣ⠨߃ࠄࠇߚ. ⴊ∥ࠍ⹜ᢱߣߒߚ႐ว, PKಣℂߦࠃࠅ, Aဳ, Bဳ, ABဳߣ߽ߦ16୚Ꮧ㉼߹ߢ㓁ᕈߩ෻ ᔕࠍ␜ߒߚ(Fig. 1-18, 19, 20). ߒߚ߇ߞߡ, ᧄᴺߦࠃࠆⴊ∥ਛߩABOᑼⴊᶧဳ᛫ේߩᬌ ಴ᗵᐲߪ, ߅߅ࠃߘ16୚Ꮧ㉼߹ߢߣ⠨߃ࠄࠇߚ. 1-4-6 ⹜ᢱߩ೨ಣℂ ᧄᴺࠍᔕ↪ߒߡABOᑼⴊᶧဳᬌᩏࠍⴕ߁㓙ߦᔅⷐߥ⹜ᢱߩಽ㊂ߪ, ᓥ᧪ߩⴊᷡቇ⊛

(40)

38 ᬌᩏᴺߦᲧߴࠆߣᄙߊࠍ૶↪ߔࠆߎߣߣߥࠆ. ߒ߆ߒ, ⹜ᢱߩ೨ಣℂߣߒߡታᣉߒߡ޿ ࠆPKಣℂߣᾲಣℂߪ, DNA᛽಴ᤨߩ⚦⢩Ⲣ⸃ᶧߩ⺞⵾ߣ߶߷౒ㅢߩᎿ⒟ߢ޽ࠆߎߣ߆ ࠄ, DNAဳᬌᩏߦ↪޿ࠆ⚦⢩Ⲣ⸃ᶧࠍዋ㊂૶↪ߔࠆߎߣߢABOᑼⴊᶧဳߩᬌᩏࠍหᤨ ߦታᣉߢ߈ࠆߣ⠨߃ࠄࠇߚ. ㅢᏱ, DNA᛽಴ᤨߩPKಣℂߦߪSDS╬ߩ⇇㕙ᵴᕈ೷ࠍ૬↪ ߔࠆߚ߼, ELISAߦᓇ㗀ࠍਈ߃ߥ޿⇇㕙ᵴᕈ೷ߩㆬቯ߇น⢻ߣߥࠇ߫, ᬌ಴ᗵᐲࠍ਄ߍ ࠆߎߣ߇ߢ߈ࠆߣ߽⠨߃ࠄࠇ, ߐࠄߦᡷༀߩ૛࿾߇޽ࠆߣ⠨߃ࠄࠇࠆ. ᧄᴺߦ߅ߌࠆPKಣℂ෸߮ᾲಣℂߪ, ࠨ࡯ࡕࡒࠠࠨ࡯ࠍ೑↪ߔࠆߎߣߢⶄ㔀ߥᠲ૞ࠍ ᔅⷐߣߖߕߦ◲ଢߦታᣉߢ߈ࠆ. PKಣℂ෸߮ᾲಣℂࠍណ↪ߒߚߎߣߦࠃࠅ, ⴊᶧ⹜ᢱ߆ ࠄ߽ABOᑼⴊᶧဳ᛫ේߩᬌ಴߇น⢻ߣߥߞߚ. 1-4-7 ߹ߣ߼ ⹜ᢱߩ೨ಣℂࠍᡷༀߒ, 㑆ធ┹วᴺߦࠃࠆELISAࠍᔕ↪ߔࠆߎߣߢ, ᓥ᧪࿎㔍ߣߐࠇ ߡ޿ߚⴊ∥ߦߟ޿ߡ߽᛫ේߩᬌ಴߇น⢻ߣߥࠅ, ⹜ᢱߩ⒳㘃ߦࠃߞߡᬌᩏᣇᴺࠍᄌᦝ ߔࠆߎߣߥߊ, ৻ᐲߦABOᑼⴊᶧဳ᛫ේߩᬌ಴߇น⢻ߣߥߞߚ. ᧄᚻᴺߪ, ⹜ᢱߩ⒳㘃 ߦ㑐ࠊࠄߕ᛫ේߩᬌ಴߇น⢻ߢ޽ࠆߎߣ, ELISAࠍၮᧄࡈࠜ࡯ࡑ࠶࠻ߦߒߡ޿ࠆߎߣ߆ ࠄ, ࡑࠗࠢࡠࡊ࡟࡯࠻࠙ࠜ࠶ࠪࡖ࡯߿ࡑ࡞࠴࠴ࡖࡦࡀ࡞ࡇࡍ࠶࠻ࠍᵴ↪ߔࠆߎߣߢᄙ ߊߩᠲ૞ߩᾘ㔀ߐࠍ⥄േൻߢ߈, ᠲ૞ᚻᴺߩ⠌ᾫߩ࿎㔍ߐ߽シᷫߔࠆߎߣ߇ߢ߈ࠆ. ߹ ߚ, ᄙᢙߩ⹜ᢱࠍหᤨߦಽᨆߔࠆߎߣ߇น⢻ߥߎߣ߆ࠄ, ᧲ᣣᧄᄢ㔡ἴߩࠃ߁ߥᄙߊߩ ᱫ⠪, ⴕᣇਇ᣿⠪ࠍ⊒↢ߐߖࠆᄢⷙᮨἴኂߦ߅ߌࠆりర⏕⹺૞ᬺᤨߦ, DNAဳߦࠃࠆり ర⏕⹺૞ᬺߣߣ߽ߦ৻ᰴࠬࠢ࡝࡯࠾ࡦࠣᴺ߿◲ᤃᬌᩏߣߒߡᄙᢙߩ⹜ᢱ߆ࠄABOᑼⴊ ᶧဳࠍ৻ᐲߦᬌᩏߒߚ޿႐วߥߤߦᄢ߈ߥ⽸₂߇ߢ߈ࠆߣ⠨߃ࠄࠇࠆ.

(41)

39 ╙㧞┨ ABOᑼⴊᶧဳᬌᩏߩ⺋್ቯߦ㑐ਈߔࠆ࿯ფᓸ↢‛ 2-1 ✜⸒ ᄢⷙᮨἴኂᤨߦ߅ߌࠆりర⏕⹺߇ᔅⷐߥᬌ૕ߩᄙߊߪ, ደᄖߦㆮ⇐ߐࠇߡ޿ࠆߎߣ ߇ᄙ޿. ߎࠇࠄߪ, ᳇᷷, Ḩᐲ, ᳪᨴߣ޿ߞߚⅣႺߩᓇ㗀ࠍฃߌ, ᤨ㑆ߩ⚻ㆊߣ౒ߦ, ⣣ ᢌ߇ㅴߺ, ᦭ᯏ‛ߪᓸ↢‛ߦࠃࠅಽ⸃ߐࠇࠆ. ⊒⷗߇ㆃࠇࠆ߶ߤ, ⴊᶧဳ್ቯߦㆡߒߚ ᬌ૕ߩណข߇࿎㔍ߣߥࠅ, ⴊᶧဳ᛫ේߩᬌ಴߇࿎㔍, ޽ࠆ޿ߪ⺋ߞߚဳ߇ᬌ಴ߐࠇࠆߎ ߣ߇޽ࠆ. ೨ㅀߩߣ߅ࠅ, ৻ㇱߩ࿯ფᓸ↢‛߇ᜬߟᄙ♧㘃߿♧࠲ࡦࡄࠢ㘃߇ABOᑼⴊᶧ ဳ᛫ේࠍ᦭ߔࠆߎߣ߇⍮ࠄࠇߡ޿ࠆ35)_{. ߹ߚ, ᓸ↢‛ߦࠃߞߡ, ABOᑼⴊᶧဳ᛫ේ߇ಽ} ⸃ߐࠇᧄ᧪ߩⴊᶧဳ߇ᬌ಴ߢ߈ߥߊߥࠆߎߣ߽⍮ࠄࠇߡ޿ࠆ1, 13, 14, 15)_{. ߎࠇࠄߪ, ᓸ↢} ‛ߩ᦭ߔࠆglycosidaseߦࠃࠆ߽ߩߣ⠨߃ࠄࠇߡ޿ࠆ. ↢૕↱᧪ߩ᦭ᯏ‛ࠍಽ⸃ߒߡ޿ࠆ࿯ფᓸ↢‛ߩਛߦߪ, ABOᑼⴊᶧဳ᛫ේಽ⸃ᵴᕈࠍ ᜬߟglycosidaseࠍ᦭ߔࠆᓸ↢‛ߩሽ࿷߇੍ᗐߐࠇࠆ߇, ߘࠇࠄߦߟ޿ߡߩᬌ⸛ߪ, ޽߹ ࠅㅴዷ߇ߥ޿. ߘߎߢ, ࡅ࠻ⴊᶧࠍ໑৻ߩᚑಽߣߔࠆኙᄤၭ࿾ࠍ↪޿ߡ, ࿯ფ߆ࠄᓸ↢‛ࠍන㔌ߒߡ, ߘࠇࠄߩਛߦglycosidaseᵴᕈࠍᜬߟᓸ↢‛߇ߤࠇ߶ߤሽ࿷ߔࠆߩ߆ࠍ⺞ᩏߔࠆߎߣߣ ߒ ߚ. ߪ ߓ ߼ ߦ , ⴊ ᶧ ဳ ᛫ ේ ߩ ᧃ ┵ ߦ ૏ ⟎ ߔ ࠆ GalNAc, Gal, Fuc ࠍ ಾ ᢿ ߔ ࠆ D-N-acetylgalactosaminidase, D-galactosidase, D-fucosidaseᵴᕈ᷹ቯࠍታᣉߔࠆߎߣߣߒߚ. ᰴߦ, glycosidaseᵴᕈࠍ᦭ߔࠆ⩶ᩣߦߟ޿ߡ, ↢૕⾗ᢱਛߩ᛫ේࠍಽ⸃น⢻ߢ޽ࠆ߆, ╙৻┨ߢ㐿⊒ߒߚ㑆ធ┹วELISAࠍᔕ↪ߒߡ, ABOᑼⴊᶧဳ᛫ේߩಽ⸃ࠍ⹜ߺߚ.

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40 2-2 ታ㛎᧚ᢱ෸߮ታ㛎ᣇᴺ

2-2-1 ታ㛎᧚ᢱ

4-nitrophenyl N-acetyl-D-D-galactosaminide(pNP-DGalNAc),

4-nitrophenyl-D-D-galactopyranoside(pNP-DGal),

4-nitrophenyl-D-L-fucopyranoside(pNP-DFuc) ߅ࠃ߮4-nitrophenolߪ, Sigma␠ࠃࠅ⾼౉ߒ ߚ. 㑆ធ┹วELISAߦ↪޿ߚ⹜⮎ߪ╙৻┨ߩታ㛎ߣหߓ߽ߩࠍ૶↪ߒߚ. 2-2-2 ࿯ფߩណข ጘ㒂⋵ౝ෸߮ߘߩㄭ㇠58ࠞᚲࠃࠅ, Ṍ⩶ߒߚࠬࡄ࡯࠹࡞ߣ50 mlኈߩṌ⩶ᷣߩࡊ࡜ࠬ ࠴࠶ࠢ⵾㆙ᔃ▤ࠍ↪޿ߡ࿯ფࠨࡦࡊ࡞ࠍណขߒߚ. 2-2-3 ⩶ᩣߩಽ㔌 ࿯ფࠨࡦࡊ࡞1 gࠍṌ⩶ߒߚ↢ℂ⊛㘩Ⴎ᳓1 ml (0. 85% NaCl)ߦ❐Ộߒߡ10୚ㅪ⛯Ბ㓏 Ꮧ㉼ߒ, Ꮧ㉼ᶧ100 μlࠍࡅ࠻ⴊᶧኙᄤၭ࿾ߦႣઃߒߡ, 25 ºCߢ48ᤨ㑆ၭ㙃ߒߚ. ߥ߅, ࡅ ࠻ⴊᶧኙᄤၭ࿾ߪ, ⟠ⴊᶧኙᄤၭ࿾ߩ⺞⵾ᴺࠍෳ⠨42)_{ߦᣂߚߦ⠨᩺ߒߚ. ߔߥࠊߜ, ↢} ℂ⊛㘩Ⴎ᳓෸߮1. 5 %㊂ߩኙᄤࠍࠝ࡯࠻ࠢ࡟࡯ࡉṌ⩶ᓟ, ቶ᷷ߢ᡼⟎ߒߡ⚂60ºC߹ߢ಄ ߿ߒ, ⋥ߜߦᧂṌ⩶ߩࡅ࠻ABဳⴊᶧ(ోⴊ)ࠍ5 %ߦߥࠆࠃ߁ߦή⩶⊛ߦട߃, Ṍ⩶ࠪࡖ ࡯࡟ߦಽᵈߒߡ࿕ൻߐߖߡ૞ᚑߒߚ. ಴⃻ߒߚࠦࡠ࠾࡯ࠍ⦡⺞, ࠨࠗ࠭, ṁⴊ෻ᔕߩ᦭ήࠍᜰᮡߣߒߡ࡜ࡦ࠳ࡓߦㆬᛮߒ, หၭ࿾ߦࠃࠆၭ㙃ࠍⶄᢙ࿁➅ࠅ㄰ߒߡଏਈ⩶ᩣߣߒߚ. ߥ߅, ৻ㅪߩᠲ૞ߪή⩶⊛ߦⴕߞߚ. 2-2-4 D-glycosidaseᵴᕈ᷹ቯ

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41

Fucࠍ᦭ߔࠆpNP-DGalNAc, pNP-DGal߅ࠃ߮pNP-DFucࠍߘࠇߙࠇၮ⾰ߣߒߡ೑↪ߒ, D-N-acetylgalactosaminidase, D-galactosidase, D-fucosidaseᵴᕈ᷹ቯࠍⴕߞߚ. ߔߥࠊߜ, ଏਈ⩶ᩣ380ᩣߦߟ޿ߡ, ߘࠇߙࠇLBၭ࿾3 mlࠍ↪޿ߡ, 25ºCߢ48ᤨ㑆ၭ㙃ᓟ, ㆙ᔃಽ㔌 (25,000 ×g 10ಽ)ߒߡ⩶૕ߣၭ㙃਄ᷡߦಽ㔌ߒߚ. ಽ㔌ߒߚ⩶૕ߪ200 μlߩ0. 15 M Phosphate buffered saline (PBS, pH 7. 5)ߦ Ộߒߡ, ᶧ૕⓸⚛ࠍ↪޿ߚಓ⚿Ⲣ⸃ಣℂࠍⴕ ޿, ၭ㙃਄ᷡߣಓ⚿Ⲣ⸃ಣℂᓟߩ⩶૕᛽಴ᶧࠍ෻ᔕᶧߣߒߚ.

pNP-DGalNAc, pNP-DGal, pNP-DFuc (޿ߕࠇ߽Ớᐲ0. 125 mM 0. 15M PBS pH 7. 5ߦṁ ⸃)ߘࠇߙࠇ40 μlࠍ96ⓣࡊ࡟࡯࠻ߦಽᵈᓟ, ၭ㙃਄ᷡ߹ߚߪ⩶૕᛽಴ᶧࠍ10 μlࠍᷙวߒ, 37 ºCߢ1ᤨ㑆෻ᔕߐߖߚ. ෻ᔕᓟ, 0. 5 M὇㉄࠽࠻࡝࠙ࡓṁᶧ50 μlࠍട߃ߡ෻ᔕࠍ஗ᱛ ߐߖ, ㆆ㔌ߒߚ4-nitrophenolߩๆశᐲ(405 nm)ࠍ, ࡊ࡟࡯࠻࡝࡯࠳࡯ࠍ↪޿ߡ᷹ቯߒߚ. ᬌ㊂✢ߪ, 0. 125 mMߩ4-Nitrophenolࠍ2୚Ბ㓏Ꮧ㉼ߒߡ૞ᚑߒ, ࠲ࡦࡄࠢ⾰Ớᐲߪ, BSA ࠍၮḰߣߒߚࡉ࡜࠶࠼ࡈࠜ࡯࠼ᴺߦࠃࠅ᷹ቯߒߚ. 2-2-5 㑆ធ┹วELISAߦࠃࠆBဳ᛫ේಽ⸃ᵴᕈ᷹ቯ D–galactosidaseᵴᕈࠍ⹺߼ߚ13⩶ᩣߪ, Bဳ᛫ේߩᧃ┵ߦሽ࿷ߔࠆDGalࠍಾᢿߔࠆน ⢻ᕈ߇㜞޿ߣ⠨߃ࠄࠇߚߎߣ߆ࠄ, ߎࠇࠄ13⩶ᩣߦߟ޿ߡ, 㑆ធ┹วELISAߦࠃࠆBဳ ᛫ේߩಽ⸃ᵴᕈ᷹ቯࠍⴕߞߚ ┹ว᛫ේߣߒߡ, Bဳߩࡅ࠻ໜᶧࠍ೑↪ߒߚ. หᗧࠍᓧߡBဳ࡮ಽᴲဳߩੱ‛߆ࠄ๺⚕ ߦໜᶧࠍណขߒ, ቶ᷷ߢੇ῎ߐߖߡ෼㓸ߒߚ. ෼㓸ߒߚ๺⚕ (20ੱಽ)ࠍ⚦ಾߒߡ, 20 ml ߩPBSߢ᛽಴ߒ, 20 ಽ㑆ᴣ㛛᳓ਛߢᾲಣℂߒߡ㒰࠲ࡦࡄࠢߣໜᶧ㉂⚛ߩᄬᵴൻࠍ ⴕ޿, ⛯޿ߡㅘᨆ࠴ࡘ࡯ࡉࠍ↪޿ߡỚ❗ߒߡ, 1 mlߩBဳໜᶧ᛽಴‛ࠍᓧߚ. ࡉ࡜࠶࠼ࡈ ࠜ࡯࠼ᴺߦࠃࠆ࠲ࡦࡄࠢ⾰Ớᐲߪ, 0. 177 μg/mlߢ޽ߞߚ. D-galactosidaseᵴᕈࠍ⹺߼ߚ13⩶ᩣߦߟ޿ߡ, ೨ၭ㙃(LBၭ࿾3 ml, 25 ºC)12ᤨ㑆, ᧄၭ 㙃(LBၭ࿾100 ml, 25 ºC) 48ᤨ㑆ࠍⴕ޿, ⩶૕ࠍ㆙ᔃಽ㔌(25,000 ×g 10ಽ)ߒߡ࿁෼ᓟ, pH

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42 6.0ߩ࡝ࡦ㉄ࡃ࠶ࡈࠔ࡯ߦ Ộ, ㆙ᔃಽ㔌ߩᠲ૞ࠍ3࿁➅ࠅ㄰ߒߡ⩶૕ࠍᵞᵺߒߡ, ELISAࠍⴕ߁߹ߢ-80 ºCߢ଻ሽߒߚ. ᓧࠄࠇߚ⩶૕(ḨẢ㊀㊂ߢ⚂1 g)ࠍ੃㋬਄ߢᶧ૕⓸ ⚛ࠍㆡቱട߃ಓ⚿ߐߖߡߔࠅߟ߱ߒ, pH6. 0ߩ࡝ࡦ㉄ࡃ࠶ࡈࠔ࡯1 mlࠍട߃ߡṁ⸃, 37 ºCߩࠗࡦࠠࡘࡌ࡯࠲࡯ౝߢࠪࡖ࡯ࡌ࠶࠻⁁ߦߥࠆ߹ߢട᷷ߒߡⲢ⸃ࠍⴕ޿, ಓ⚿ߣⲢ ⸃ࠍ3࿁➅ࠅ㄰ߒߚᓟ, 20ಽ㑆㆙ᔃಽ㔌(4 ºC, 25,000 ×g)ߒߡ, ਄ᷡࠍ⩶૕᛽಴ᶧߣߒߚ. ┹ว᛫ේࠍpH6. 0ߩ࡝ࡦ㉄ࡃ࠶ࡈࠔ࡯ߢ400୚߆ࠄᲑ㓏Ꮧ㉼ߒߚ߽ߩߘࠇߙࠇ15 μlߦ, ⩶૕᛽಴ᶧ15 μlࠍട߃, ᶧ㊂ࠍ30 μlߣߒߡ, 37 {Cߢ24ᤨ㑆෻ᔕߐߖߚ. ෻ᔕᓟ, 95 { Cߢ15ಽ㑆ᾲಣℂࠍⴕߞߡ, ᷙวᶧਛߩ㉂⚛ࠍᄬᵴߐߖߚ. ᾲಣℂᓟ, ৻ᰴ᛫૕ߣߒߡ ⴊᶧဳ್ቯ↪ࡑ࠙ࠬ↱᧪ࡕࡁࠢࡠ࡯࠽࡞᛫B᛫૕(PBSߢ800୚Ꮧ㉼, ࠗࡓࠦࠕ␠⵾) 30 μlࠍട߃ߡᶧ㊂ࠍ60 μl (┹ว᛫ේ - ৻ᰴ᛫૕ᷙวᶧ)ߣߒ, 4 {Cߢ12ᤨ㑆㕒⟎ߒߡ┹ว ᛫ේߦ৻ᰴ᛫૕ࠍๆ⌕ߐߖߚ. ޽ࠄ߆ߓ߼, 96ⓣࡊ࡟࡯࠻(Maxisorp, Nunc␠⵾)ߦBဳ♧㎮ࡊࡠ࡯ࡉ(1. 0 μg/ml) 50 μl ࠍಽᵈߒ, 4 ºCߦ1᥅㕒⟎ߒߡฦ࠙ࠚ࡞ߦBဳ♧㎮ࡊࡠ࡯ࡉࠍๆ⌕ߐߖ࿕⋧ൻߒߚ. ࡊ࡟ ࡯࠻ߩᵞᵺ, ੑᰴ᛫૕ߩ෻ᔕ, ⊒⦡෻ᔕߪ, ╙৻┨ߣห᭽ߦⴕߥߞߚ. ߥ߅, ৻ᰴ᛫૕ߩή޿߽ߩࠍࡉ࡜ࡦࠢߣߒ. 㒶ᕈࠦࡦ࠻ࡠ࡯࡞ߣߒߡ⩶૕᛽಴ᶧࠍᾚ ᴣ ಣ ℂ ߒ ߚ ߽ ߩ, 㓁ᕈࠦࡦ࠻ ࡠ࡯࡞ߣߒ ߡD-galactosidase (green coffee bean↱᧪, prozyme␠⵾)ࠍ5 U/mlߩỚᐲߢ↪޿ߚ.

৻ㅪߩᠲ૞ߪ, Fig. 2-1ߦ␜ߒߚ.

ߥ߅, 9-1ᩣߦߟ޿ߡ, ၭ㙃ࠍ➅ࠅ㄰ߒߡ5 g(ḨẢ㊀㊂)ߩ⩶૕ࠍᓧߡ, ห᭽ߦಓ⚿ߣⲢ ⸃ಣℂߦࠃࠅ, 5 mlߩ⩶૕᛽಴ᶧࠍ⺞ᢛߒ, ⛯޿ߡㅘᨆ࠴ࡘ࡯ࡉࠍ↪޿ߡ1 mlߦỚ❗ߒ ߡ, ⚂5୚ߩỚᐲߩ⩶૕᛽಴ᶧࠍ૞ᚑߒߡ, ห᭽ߦ㑆ធ┹วELISAߦଏߒߚ.

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43

Fig. 2-1 Schematic of theindirect competitive ELISA for the detection of B antigens. a. Crude enzymes (15 μL) were added to saliva extract (15 μL) and placed at 37 ºC for 24 hours. The reaction was stopped by boiling. b. An equal volume (30 μL) of sample was combined with anti-B MAb and incubated at 4 ºC for 12 hours. c. The wells of polystyrene ELISA plates were coated with type B (tri)-PAA in 50 μL PBS at 4 ºC for 12 hours. The wells were washed three times with 250 μL PBST. d. The wells were blocked with 100 μL BSA-PBS and incubated at 37ºC for 1 hour. At the completion of the incubation the wells were washed with 250 μL PBST three times. e. A 50μL aliquot containing crude enzyme and saliva extract combined with anti-B MAb was added to the wells. Plates were incubated at 37 ºC for 1 hour prior to washing with 250 μL PBST three times. f. Anti-mouse IgM-HRP conjugate (50 μL) was added to each well. The plates were incubated at 37ºC for 1 hour, prior to washing with 250 μL PBST three times. g. o-Phenylenediamine solution (50 μL) was added to each well, and the plates incubated at room temperature for 10 min. The colorimetric reaction was stopped by the addition of 100 μL 1M sulfuric acid to each well. The OD was measured at 490 nm using a spectrophotometer.

c

d

e _f

b

g

anti-B monoclonal antibody antigen BSA

Anti mouse IgM HRP conjugated

a

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44 2-2-6 DNA᛽಴ߣ16S rRNAㆮવሶ⸃ᨆ

⩶ᩣࠍLBၭ࿾3 mlࠍ↪޿ߡ25 {Cߢ24ᤨ㑆ၭ㙃ᓟ㧘ၭ㙃ᶧࠍ㆙ᔃಽ㔌(15000rpm, 15 min)ߒߡ⩶૕ࠍ࿁෼ߒߚ. ࿁෼ߒߚ⩶૕ࠃࠅISOIL Beads Beating(࠾࠶ࡐࡦࠫ࡯ࡦ)ࠍ૶ ↪ߒߡࡆ࡯࠭☳⎈ᴺߦࠃࠅDNAࠍ᛽಴ߒ㧘⚦⩶16SrRNAㆮવሶࠍ․⇣⊛ߦჇ᏷ߔࠆ࡙ ࠾ࡃ࡯ࠨ࡞ࡊ࡜ࠗࡑ࡯࠮࠶࠻(Eub10F: 5̉-GTTTGATCCTGGCTCA-3̉; Uni1500R: 5̉ -TACCTTGTTACGACTG-3̉)ࠍ↪޿ߡPCRࠍⴕߥߞߚ. PCRߩ᧦ઙߪ, ᾲᄌᕈ 95 ºC 10 ಽᓟ, 35ࠨࠗࠢ࡞ߩ(ᾲᄌᕈ 95 ºC 3 ⑽, ࠕ࠾࡯࡝ࡦࠣ 55 ºC 1 ಽ, િ㐳෻ᔕ 72 ºC 90 ⑽), ᦨ⚳િ㐳෻ᔕࠍ 72 ºC 10ಽ㑆ⴕߥߞߚ.

Ⴧ ᏷ ↥ ‛ ࠍPerforma® DTR Gel FiltrationCartridges (EdgeBio) ࠍ ↪ ޿ ߡ ♖ ⵾ ߒ , BigDye®Terminator v3. 1 Cycle Sequencing Kit (Applied Biosystems)ࠍ↪޿ߡࠨࠗࠢ࡞ࠪ࡯ ࠢࠛࡦࠬ෻ᔕࠍⴕ޿, Jenetic analyzer 3130xl (Applied Biosystems)ࠍ↪޿ߡࠠࡖࡇ࡜࡝࡯ 㔚᳇ᵒേߦࠃࠅ, ࠪ࡯ࠢࠛࡦࠬࠍⴕߥߞߚ. ᓧࠄࠇߚ㈩೉ࠍ↪޿ߡBLASTߦࠃࠅ, ࡎࡕ ࡠࠫ࡯ᬌ⚝ࠍⴕߞߚ.

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45 2-3 ታ㛎⚿ᨐ 2-3-1 ࿯ფߩណข ጘ㒂⋵ਅ෸߮ߘߩㄭ㇠ߩ᳃ኅ(イਅ, ᐸ), ᴡᎹᢝ, ⇌, ጊᨋ, ㆇേ႐╬58ࠞᚲ߆ࠄ࿯ფ ࠍណขߒߚ. ណข႐ᚲߪ, ․ᱶߥⅣႺࠍㆬ߫ߕ, ࿯ფࠍㆬᛯߒߚ. 2-3-2 ⩶ᩣߩಽ㔌 ࡅ࠻ⴊᶧኙᄤၭ࿾(ABဳ)ࠍ↪޿ߡၭ㙃ࠍⴕߞߚߣߎࠈ, ಴⃻ߒߚࠦࡠ࠾࡯ߪ, ♻⁁⩶, ṁⴊ෻ᔕࠍ␜ߔ߽ߩ, ṁⴊ෻ᔕࠍ␜ߒࠢ࡝ࠕ࠱࡯ࡦߦ⦡⚛ࠍ↥↢ߔࠆ߽ߩ, ṁⴊ෻ᔕࠍ ␜ߐߥ޿߽ߩ╬߇ᷙ࿷ߒߡ޿ߚ. 2-3-3 D-glycosidaseᵴᕈ᷹ቯ

ᓧ ࠄ ࠇ ߚ380 ᩣ ߦ ߟ ޿ ߡ , pNP-DGalNAc, pNP-DGal ߅ ࠃ ߮ pNP-DFuc ࠍ ↪ ޿ ߚ glycosidaseᵴᕈ᷹ቯࠍⴕߞߚ. ߘߩ⚿ᨐ, 380ᩣਛ13⩶ᩣ(1-5, 2-4, 3-5, 4-3, 9-1, 22-2, 26-2, 34-2, 34-7, 34-9, 35-2, 41-2, 55-4)ߦD-galactosidaseᵴᕈࠍ⹺߼ߚ(Fig. 2-2). ߎࠇࠄ13⩶ᩣ ߩᕈ⁁ࠍTable 2-1ߦ␜ߔ. D-galactosidaseᵴᕈࠍ⹺߼ߚ13⩶ᩣߪ, ޿ߕࠇ߽ṁⴊ෻ᔕࠍ␜ߐߕ, ࠦࡠ࠾࡯ߩᒻᘒ ቇ⊛ᕈ⁁ߣD-galactosidaseᵴᕈߣߩ⋧㑐㑐ଥߪ⹺߼ࠄࠇߥ߆ߞߚ. ߘࠇࠄߩ߁ߜ11⩶ᩣ ߩࠦࡠ࠾࡯ߩᒻ⁁ߪ౞ᒻߢ, ᱷࠆ26-2ᩣߪ♻⁁ߢ޽ߞߚ. ߹ߚ, ࿯ფߩណข႐ᚲߦ౒ㅢ ߩᚲ⷗ߪ⹺߼ࠄࠇߥ߆ߞߚ(Table 2-1). ߹ߚ, ੹࿁ㆬᛮߒߚᓸ↢‛ਛߦߪ, D-N-acetylgalactosaminidase, D–fucosidaseᵴᕈࠍ᦭ ߔࠆᓸ↢‛ߪሽ࿷ߒߥ߆ߞߚ. D-galactosidaseᵴᕈࠍ⹺߼ߚ13⩶ᩣߦߟ޿ߡ, ࡅ࠻Bဳໜᶧࠍ┹ว᛫ේߦ↪޿ߚ㑆ធ ┹วELISAߦଏߒߚ.

(48)

(49)

47

Fig. 2-1 Specific D-galactosidase activities of 13 strains using pNP-D-Gal as substrate

pNP-DGal was incubated with cell free extract (black columns) or culture supernatant (white

columns). PBS was used as negative control (NC). Galactosidase negative strain (1-1) was included in comparison. Tests were performed in duplicate. Error bars represent the S. D.

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48 2-3-4 㑆ធ┹วELISAߦࠃࠆBဳ᛫ේಽ⸃ᵴᕈ᷹ቯ D-galactosidaseᵴᕈࠍ⹺߼ߚ13⩶ᩣߪ, Bဳ᛫ේߩᧃ┵ߦሽ࿷ߔࠆDGalࠍಾᢿߔࠆน⢻ ᕈ߇㜞޿ߣ⠨߃, ߎࠇࠄߩ⩶૕᛽಴ᶧߦߟ޿ߡ, 㑆ធ┹วELISAࠍ೑↪ߒߚBဳ᛫ේߩ ಽ⸃ᵴᕈ᷹ቯࠍⴕߞߚ. ߘߩ⚿ᨐࠍFig. 2-3ߦ␜ߔ. 㓁ᕈࠦࡦ࠻ࡠ࡯࡞ߣߒߡ૶↪ߒߚ ࠦ࡯ࡅ࡯⼺↱᧪ߩD-galactosidaseߪ, ໜᶧਛߩBဳ᛫ේࠍಽ⸃ߒ, ๆశᐲ߇਄᣹ߒߚ(Fig. 2-3). D-galactosidaseᵴᕈࠍ⹺߼ߚ13⩶ᩣਛ7⩶ᩣ(2-4, 9-1, 22-2, 26-2, 34-2, 34-7, 35-2)ߪ, ᾲಣℂࠨࡦࡊ࡞ߣᲧセߒߡๆశᐲߩ਄᣹ࠍⷰኤߢ߈(Fig. 2-3a), ਛߢ߽9-1ߪᦨ߽㜞޿ᵴ ᕈࠍ␜ߒߚ(Fig. 2-3b).

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49

Fig. 2-2 Blood group B antigen degrading activities using an indirect competitive ELISA. Cell free extracts (black columns) or boiled extracts (boil control; white columns) were incubated with the competitive antigen (type B human saliva diluted 1:6400) at 37 ºC for 24 hr. Reactions were stopped by heat treatment at 95 ºC for 15 min. The quantity of B antigen was determined by the indirect competitive ELISA. PC stands for positive control (coffee bean D-galactosidase). Tests were performed in duplicate. Error bars represent the S. D.

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50 2-3-5 DNA᛽಴ߣ16S rRNAㆮવሶ⸃ᨆ

D-galactosidaseᵴᕈࠍ⹺߼ߚ13⩶ᩣ(34-2ࠍ㒰ߊ)ߩ․ᕈߪ, Table 2-1ߦ␜ߒߚߣ߅ࠅߢ, 34-2ᩣߪ, 16S rRNAㆮવሶࠍჇ᏷ߔࠆߎߣ߇ߢ߈ߥ߆ߞߚ.

16S rRNA ㆮવሶ㈩೉ߩࡎࡕࡠࠫ࡯ᬌ⚝ߩ⚿ᨐ, 1-5ᩣߪBacillus megaterium strain MC-3-SDCH4, , 2-4ᩣߪBacillus spp. SuP1ߣ, 3-5ᩣߣ22-2ᩣߪBacillus sp. WS24ߣ, 4-3ᩣߣ 34-9ᩣߪBacillus megaterium WS 19ߣ, 9-1ᩣߪBacillales bacterium TB5ߣ, 26-2ᩣߪ

Bacillus sp. SGE97(2010)ߣ, 34-7ᩣߣߪBacillus sp. S10,ߣ, 35-2ᩣߪBacillus megaterium

strain AIMST 7. M5. 2ߣ, 41-2ᩣߪBacillus thuringiensis strain S22Ba-195ߣ, 55-4ᩣߪ

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51 2-4 ⠨ኤ 2-4-1 ណข⩶ᩣ ಴⃻ߒߚࠦࡠ࠾࡯ࠍ, ⦡⺞, ࠨࠗ࠭, ṁⴊ෻ᔕߩ᦭ήࠍᜰᮡߣߒߡㆬᛮߒ, ࡅ࠻ⴊᶧ ኙᄤၭ࿾ࠍ↪޿ߡ380ᩣࠍಽ㔌ߒߚ. ࡅ࠻ⴊᶧኙᄤၭ࿾ߦߪ, ࡅ࠻ⴊᶧ෸߮NaClߩߺࠍ ᷝടߒߡ޿ࠆߎߣ߆ࠄ, ࿯ფ߆ࠄࡅ࠻ⴊᶧ↱᧪ߩᚑಽ߹ߚߪኙᄤ↱᧪ᚑಽࠍ೑↪ߒߡ ↢⢒น⢻ߥᓸ↢‛ࠍ෼㓸ߢ߈ߚߣ⠨߃ࠄࠇߚ. 2-4-2 D-glycosidaseᵴᕈ 13⩶ᩣਛ3⩶ᩣ(1-5, 3-5, 9-1)ߪ, ෻ᔕᶧਛߩpNP-DGal(5 mmol)ࠍోߡಽ⸃ߒߡ޿ࠆߣ ⠨߃ࠄࠇ, ߘߩઁߩᵴᕈࠍ᦭ߒߚ⩶ᩣߦᲧߴࠆߣ㜞޿ᵴᕈࠍ␜ߒ(Fig. 2-1), Bဳ᛫ේߩ ಽ ⸃ ᩣ ߣ ߒ ߡ ᦭ ജ ߥ ୥ ⵬ ߣ ⠨ ߃ ࠄ ࠇ ߚ. Liu ࠄ ߪ , 2500 ᩣ ߩ න 㔌 ᩣ ࠍ ↪ ޿ ߡ , 7-amino-4-methyl-coumarin labeled B tetrasaccharideࠍၮ⾰ߣߔࠆD-galactosidaseᵴᕈࠍ⺞ ᩏߒ, 5ᩣߦD-galactosidaseᵴᕈࠍᬌ಴ߒߡ޿ࠆ߇, pNP-DGalߩಽ⸃⩶ߪᬌ಴ߒߡ޿ߥ޿ 24)_. ߹ߚ, ੹࿁ㆬᛮߒߚᓸ↢‛ਛߦߪ, D-N-acetylgalactosaminidaseᵴᕈࠍ␜ߔᓸ↢‛ߪሽ ࿷ߒߥ߆ߞߚ. Liu ࠄߩႎ๔ߢ߽, D-N-acetylgalactosaminidaseᵴᕈࠍ᦭ߔࠆᓸ↢‛ߪᬌ ಴ߐࠇߡ޿ߥ޿24). ߐࠄߦ, ᧄታ㛎ߢߪD–fucosidaseᵴᕈࠍ᦭ߔࠆᓸ↢‛ߪ⏕⹺ߢ߈ߥ ߆ߞߚ. 2-4-3 㑆ធ┹วELISAߦࠃࠆBဳ᛫ේಽ⸃ᵴᕈ ╙৻┨ߢㅀߴߚ㑆ធ┹วELISAࠍ↪޿ߡ, D-galactosidaseᵴᕈࠍ᦭ߒߡ޿ߚ13⩶ᩣߩB ဳ᛫ේಽ⸃ᵴᕈߩ᷹ቯࠍⴕߞߚ. ┹ว᛫ේߪ, ታ㓙ߩ↢૕⹜ᢱߢ޽ࠆBဳߩࡅ࠻ໜᶧࠍ ↪޿, ↢૕ਛߦሽ࿷ߔࠆABOᑼⴊᶧဳ᛫ේߣ⩶૕᛽಴ᶧߣߩ෻ᔕߦࠃࠆBဳ᛫ේߩᷫዋ ࠍ, 㑆ធ┹วELISAߦࠃࠅ᷹ቯߒߚ.

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52 D-galactosidaseᵴᕈࠍ᦭ߒߡ޿ߚ13⩶ᩣߩ߁ߜ㧣⩶ᩣ(2-4, 9-1, 22-2, 26-2, 34-2, 34-7, 35-2)ߩ ⩶૕᛽಴ᶧߣ㓁ᕈࠦࡦ࠻ࡠ࡯࡞ߣߒߡ૶↪ߒߚࠦ࡯ࡅ࡯⼺↱᧪ߩD-galactosidaseߪ, B/B0(%) ߩ୯߇ᾚᴣࠨࡦࡊ࡞㧔㒶ᕈࠦࡦ࠻ࡠ࡯࡞㧕ߣᲧߴ, 㜞޿୯ࠍ␜ߒ, Bဳ᛫ේ߇ಽ⸃ߐࠇߡ޿ࠆ ߎߣࠍ␜ߒߡ޿ࠆ (Fig. 2-2). ߘࠇࠄߩ߁ߜᦨ߽㜞޿Bဳ᛫ේಽ⸃ᵴᕈࠍ␜ߒߚ9-1ᩣߪ, ⩶૕㊂ࠍ5୚ߦჇᄢߔࠆߎߣߢ, 㓁ᕈࠦࡦ࠻ࡠ࡯࡞ߣห⒟ᐲߩಽ⸃ᵴᕈࠍ␜ߒߚߎߣ߆ࠄ, Bဳ᛫ේಽ⸃ᵴᕈߪ⩶૕㊂ߦଐሽ ߒߡ޿ࠆߎߣ߇ࠊ߆ߞߚ. ߹ߚ, 9-1ᩣߪpNP-DGalࠍၮ⾰ߣߔࠆD-galactosidaseᵴᕈ߽㜞޿୯ࠍ␜ߒߡ޿ࠆߎߣ߆ࠄ (Fig. 2-1), 㑆ធ┹วELISAߦ߅޿ߡⷰኤߐࠇߚBဳ᛫ේߩಽ⸃ᵴᕈߪ, Bဳ᛫ේߩᧃ┵ߦሽ࿷ ߔࠆGalࠍಾᢿߔࠆߎߣߦࠃࠆߣ⠨߃ࠄࠇߚ. 2-4-4 DNA᛽಴ߣ16S rRNAㆮવሶ⸃ᨆ 16S rRNA ㆮવሶ㈩೉ߩࡎࡕࡠࠫ࡯ᬌ⚝ߩ⚿ᨐ, galactosidaseᵴᕈࠍ᦭ߒߡ޿ߚ12⩶ᩣ ߪ޿ߕࠇ߽Bacillusዻߦ㕖Ᏹߦㄭ✼ߥᩣߢ޽ߞߚ. ߎࠇ߹ߢߦ, Bဳ᛫ේಽ⸃⚦⩶ߣߒߡ

⍮ࠄࠇߡ޿ࠆᓸ↢‛߇Bacillus cereusߢ޽ߞߚ13)_{ߎߣ߆ࠄ߽, galactosidaseᵴᕈߣBacillus}

ዻߦߪ, ⋧㑐㑐ଥ߇޽ࠆߎߣ߇␜ໂߐࠇߚ.

1-5ᩣߪ, B. megaterium strain MC-3-SDCH4ߣ99㧑એ਄ߩ⋧หᕈࠍ␜ߒ, B. megaterium

strain MC-3-SDCH4ߪ, ㋶‛ߩ㘑ൻ߿࿯ფᒻᚑࠍᜂߞߡ޿ࠆߣႎ๔ߐࠇߡ޿ࠆ10㧕_.

3-5ᩣߣ22-2ᩣߪ, Bacillus sp. WS24ߣ99㧑ߩ⋧หᕈࠍ␜ߒ, Bacillus sp. WS24ߪ, ዊ㤈

ߩᩮ࿤߆ࠄන㔌ߐࠇߡ޿ࠆ64)_.

2-4ᩣ߅ࠃ߮34-7ᩣߪ, Bacillus sp. SuP1߅ࠃ߮S10ߣߘࠇߙࠇ99㧑એ਄ߩ⋧หᕈࠍ␜ߒ

ߚ. 4-3ᩣ߅ࠃ߮34-9ᩣߪ, B. megaterium WS19ߣ99㧑એ਄ߩ⋧หᕈ ࠍ␜ߒߚ. B.

megaterium WS19, Bacillus sp. SuP1߅ࠃ߮S10ߪBacillussp. WS24ߣ99㧑એ਄ߩ⋧หᕈࠍ

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53

9-1ᩣߪBacillales bacterium TB5ߣ99㧑એ਄ߩ⋧หᕈࠍ␜ߒߚ. Bacillales bacterium TB5 ߪ, ࡇ࡯࠽࠶࠷ߩᩮ࿤ࠃࠅන㔌ߐࠇߡ޿ࠆ.

26-2ᩣ߅ࠃ߮55-4ᩣߪ, Bacillus spp. SGE97(2010)߅ࠃ߮ SGE139(2010)ߣߘࠇߙࠇ 99㧑એ਄ߩ⋧หᕈࠍ␜ߒߚ. Bacillus spp. SGE97(2010)߅ࠃ߮ SGE139(2010)ߪ, B.

megaterium strain zjzl-1ߣ99㧑એ਄ߩ⋧หᕈࠍ␜ߒ, B. megaterium strain zjzl-1ߪ, ✢⯻ߦ

Ქᕈࠍ␜ߔߎߣ߇⍮ࠄࠇߡ޿ࠆ41).

35-2ᩣߪ, B. megaterium strain AIMST 7. M5. 2ߣ99㧑એ਄ߩ⋧หᕈࠍ␜ߒ, B.

megaterium strain AIMST 7. M5. 2ߪ, B. megaterium strain EL-16 ߣ99%એ਄ߩ⋧หᕈࠍ␜

ߒߚ.

41-2ᩣߪB. thuringiensis strain S22Ba-195ߣ99㧑એ਄ߩ⋧หᕈࠍ␜ߒ, B. thuringiensis strain S22Ba-195 ߪ , B. megaterium strain EL-12 ߣ 99 㧑 એ ਄ ߩ ⋧ ห ᕈ ࠍ ␜ ߒ ߚ . B.

megaterium strains EL-12 and EL-16ߪ, ᦺ㞲ੱෳߩᩮᐙࠃࠅන㔌ߐࠇߡ޿ࠆ52)_.

ߎࠇࠄߩ16S rRNAㆮવሶߩ⋧หᕈ⸃ᨆ߆ࠄ, ᧄታ㛎ߢන㔌ߒߚD-galactosidaseᵴᕈࠍ ᦭ߔࠆᓸ↢‛ߪ, ᬀ‛ߩᩮ࿤߿ᬀ‛૕ౝߦሽ࿷ߔࠆBacillusዻߩᓸ↢‛ߦ㕖Ᏹߦ㘃ૃߒ ߚᩣߢ޽ࠆߎߣ߇᣿ࠄ߆ߣߥࠅ, ߎࠇࠄߩ↥↢ߔࠆD-galactosidase߇⥄ὼⅣႺਛߩᬀ‛ ߩಽ⸃߿ᩮ࿤ߩᒻᚑࠍᜂߞߡ޿ࠆߩߢߪߥ޿߆ߣ⠨߃ࠄࠇߚ. 2-4-5 ߹ߣ߼ ੹࿁ߩታ㛎ߦࠃߞߡ, ABOᑼⴊᶧဳ᛫ේ(Bဳ᛫ේ)߇, ޿ߊߟ߆ߩBacillusዻ⚦⩶(ᓧߦ 9-1ᩣ)ߦࠃࠅಽ⸃ߐࠇࠆߎߣ߇␜ߐࠇ, ߘࠇࠄ߇ᬀ‛߿ߘߩᩮ࿤ߣᷓ޿㑐ࠊࠅ߇޽ࠆߎ ߣ߇␜ໂߐࠇߚ. ߹ߚ, ੹࿁ㆬᛮߒߚᓸ↢‛ਛߦߪ, D-N-acetylgalactosaminidaseᵴᕈࠍ␜ߔᓸ↢‛ߪሽ ࿷ߒߥ߆ߞߚ. Liu ࠄߩႎ๔ߢ߽, D-N-acetylgalactosaminidaseᵴᕈࠍ᦭ߔࠆᓸ↢‛ߪᬌ಴ ߐࠇߡ޿ߥ޿24)_{. ߐࠄߦ, ᧄታ㛎ߢߪD–fucosidaseᵴᕈࠍ᦭ߔࠆᓸ↢‛ߪ⏕⹺ߢ߈ߥ߆}

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54 ߞߚ. ߎࠇࠄߩߎߣ߆ࠄ, A෸߮Hဳ᛫ේߪBဳ᛫ේߦᲧߴ, ቟ቯߥ᛫ේߢ޽ࠆߣ߽޿߃ ࠆ. ⚿⺰ߣߒߡ, ⅣႺਛߦㆮ⇐ߐࠇߚᬌ૕ߩABOᑼⴊᶧဳࠍᬌᩏߔࠆ㓙ߦߪ, Bဳ߆ࠄO ဳ߳ߩᄌ឵ߩน⢻ᕈ߇޽ࠆߎߣߦᵈᗧࠍᛄ߁ᔅⷐ߇޽ࠅ, ⋧ᒰߥᦼ㑆, ᬀ‛߇ሽ࿷ߔࠆ ࠃ߁ߥ႐ᚲߦㆮ⇐ߐࠇߡ޿ߚ႐วߦߪ, Oဳߣ್ቯߔࠆ೨ߦᬌᩏߩ⺋್ቯࠍ⇼߁ᔅⷐ߇ ޽ࠆ.

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55 ⚳┨ ✚ว⠨ኤ 3-1-1 ↢૕⹜ᢱߩᓸ↢‛ᳪᨴ ደᄖߦㆮ⇐ߐࠇߚ⹜ᢱ߆ࠄABOᑼⴊᶧဳ᛫ේࠍᬌ಴ߔࠆ㓙, ᧄ᧪ߩⴊᶧဳߣߪ⇣ߥ ࠆဳ޽ࠆ޿ߪ್ቯ߇࿎㔍ߦߥࠆߎߣߪ, ߒ߫ߒ߫⚻㛎ߐࠇߡ߈ߚ. ߹ߚ, ᓸ↢‛ߦࠃࠆ ABOᑼⴊᶧဳ᛫ේߩಽ⸃ߦߟ޿ߡ߽, ⎇ⓥ߇ߥߐࠇߡ߈ߚ12, 13, 14, 15, 63)_{. ߒ߆ߒ, ߎࠇࠄ} ߩ⎇ⓥߪᢔ⊒⊛ߢ, ⛮⛯⊛ߦታᣉߐࠇߡ߅ࠄߕ, ಽ⸃ᵴᕈࠍᜬߟᓸ↢‛߇ሽ࿷ߔࠆߣ޿ ߁੐ታߛߌ߇, ᩮ᜚ߣߥߞߡ޿ߚ. ߐࠄߦㄭᐕߪ, DNAဳߦࠃࠆ୘ੱ⼂೎ᴺ߇᥉෸ߒߚߎߣ߆ࠄ, ABOᑼⴊᶧဳࠍᜰᮡߣ ߔࠆりర⏕⹺ߩᯏળߪᷫዋߒߡ߅ࠅ, ⺋್ቯߩේ࿃ߣߥࠆᓸ↢‛ߦߟ޿ߡ, Ⓧᭂ⊛ߥ⎇ ⓥ߇ߥߐࠇߡ޿ߥ޿⃻⁁ߢ޽ߞߚ. ߒ߆ߒ, 㜞ᐲߦ⣣ᢌߒߚᱫ૕ߢߪ, DNAߩᬌ಴߇࿎㔍ߦߥࠆ଀߽޽ࠅ, 㛽߿ᱤߥߤᱷ ሽߔࠆ⹜ᢱ߆ࠄᬌ಴ߢ߈ࠆㆮવᒻ⾰߇DNAဳߣABOᑼⴊᶧဳߛߌߣߥࠆߎߣ߽ᗐቯߐ ࠇࠆ. ೨ㅀߩࠃ߁ߦ, ᣣᧄߩࠃ߁ߦABOᑼⴊᶧဳ߇㜞ᐲߦ᥉෸ߒߚ␠ળߢߪ, ᄙᢙߩり రਇ᣿⠪ߩ࡝ࠬ࠻߆ࠄ⹥ᒰ⠪ࠍ⛉ࠅㄟ߻ߣ޿߁ὐߢ, ㊀ⷐߥㆮવ⊛ᜰᮡߣߥࠆ25, 46)_. ᧄ⎇ⓥߢන㔌ߒߚBဳ᛫ේಽ⸃ᩣߪ, ޿ߕࠇ߽Bacillusዻߦዻߔࠆᩣߢ޽ߞߚ. ᧄ⎇ⓥ એ೨ߦႎ๔ߐࠇߡ޿ࠆBဳ᛫ේಽ⸃⩶߽, Bacillus cereus13)_{ߢ޽ࠅ, ⥄ὼ⇇ߢ⿠ߎࠆBဳ᛫} ේߩಽ⸃ߦ߽, Bacillusዻߩᓸ↢‛߇㑐ਈߒߡ޿ࠆߣ⠨߃ࠄࠇࠆ. Oဳࠍ್ቯߔࠆ㓙ߦߪ, Bacillusዻ⚦⩶ߦࠃࠆᳪᨴ߿D-galactosidaseᵴᕈߩ᦭ήࠍ⏕߆߼ࠆߥߤ, ⺋್ቯߩෂ㒾ᕈ ࠍలಽߦ⠨ᘦߒ, ᧄ᧪ߪBဳߢ޽ࠆน⢻ᕈࠍలಽߦ⠨ᘦߔࠆᔅⷐ߇޽ࠆ. ߹ߚ, ᧄ⎇ⓥߢන㔌ߒߚBဳ᛫ේಽ⸃ᩣߪ, ޿ߕࠇ߽ᬀ‛߿ߘߩᩮ࿤ߦ↢ᕷߔࠆ Bacillusዻ⚦⩶ߢ޽ࠆߎߣ߇᣿ࠄ߆ߣߥߞߚ. ߎߩߎߣ߆ࠄ, ᬀ‛߇⥄↢ߒߡ޿ࠆጊ, ↰ ⇌, ᴡᎹᢝߥߤߩ⸃᡼ߐࠇߚ႐ᚲߦㆮ⇐ߐࠇߚ↢૕⹜ᢱ߇, ⺋್ቯࠍᒁ߈⿠ߎߔน⢻ᕈ ߇㜞޿ߎߣࠍᗧ๧ߒ, ᬀ‛ߩ⥄↢ߒߥ޿ㆇേ႐ߥߤߩᢛ஻ߐࠇߚ࿯ფߢߪ, Bဳ᛫ේߩ ಽ⸃ߩน⢻ᕈ߇ૐ޿ߣ⠨߃ࠄࠇߚ.

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56 3-1-2 ⅣႺᓸ↢‛↱᧪ߩ♧ട᳓ಽ⸃㉂⚛ ᧄ⎇ⓥߢߪ, 380⩶ᩣਛ13⩶ᩣߦpNP-DGalࠍၮ⾰ߣߔࠆD-galactosidaseᵴᕈࠍ⹺߼ߚ. Liu ࠄߩႎ๔ߢߪ, pNP-DGalࠍಽ⸃ߔࠆᓸ↢‛ߪᬌ಴ߐࠇߡ޿ߥ޿24)_{. ߎߩᵴᕈߩᏅ} ߪ, ၭ 㙃 ᧦ ઙ ߿ ၮ ⾰ ߩ ㆑ ޿ ߥ ߤ ߇ ේ ࿃ ߣ ⠨ ߃ ࠄ ࠇ , ߹ ߚ , Liu ࠄ ߩ ႎ ๔ ߒ ߚ D-galactosidaseߣ, ᧄ⎇ⓥߢ⷗಴ߒߚ9-1ᩣߢߪ, ㉂⚛ߩၮ⾰․⇣ᕈ߇⇣ߥࠆߣ⠨߃ࠄࠇ ࠆ. ᧄ⎇ⓥߢߪ, D-N-acetylgalactosaminidase߅ࠃ߮D-fucosidaseᵴᕈࠍ᦭ߔࠆᓸ↢‛ߪᬌ಴ ߢ߈ߥ߆ߞߚ. Liu ࠄߩႎ๔ߢ߽, D-N-acetylgalactosaminidaseᵴᕈࠍ᦭ߔࠆᓸ↢‛ߪᬌ ಴ߐࠇߡ޿ߥ޿24)_{ߎߣ߆ࠄ, N-acetylgalactosaminidase߅ࠃ߮D-fucosidaseߩሽ࿷ߪ, 㕖Ᏹ} ߦ⒘ߢ޽ࠆߣ⠨߃ࠄࠇࠆ. 3-1-3 ABOᑼⴊᶧဳ᛫ේߩඨቯ㊂⊛ಽᨆᴺ ╙৻┨ߢㅀߴߚ㑆ធ┹วELISAߪ, ๆశᐲ߇᷹ቯኻ⽎᛫ේߩ㊂ߦᲧ଀ߒߡ޿ࠆߎߣ߆ ࠄ, ᓥ᧪ߩⴊᷡቇ⊛ᚻᴺ߇㓁ᕈ߆㒶ᕈߢ޽ࠆ߆ࠍ್ቯߔࠆߩߦᲧߴ, ᛫ේߩ㊂ࠍቯ㊂⊛ ߦᲧセߔࠆߎߣ߇น⢻ߣߥߞߚ.ߎߩ㑆ធ┹วELISAࠍᔕ↪ߔࠇ߫, ᓸ↢‛ߛߌߢߥߊ, ߘߩઁߩⷐ࿃ߦࠃࠆ᛫ේߩಽ⸃߽⹏ଔߔࠆߎߣ߇ߢ߈, ੹ᓟߩⴊᶧဳ㐓ቯߩ♖ᐲะ਄ ߦߟߥߍࠆߎߣ߇ߢ߈ࠆߣ⠨߃ࠄࠇࠆ. ߹ߚ, ᧄታ㛎ߢߪAဳ, Hဳ᛫ේߩಽ⸃⩶ߩ⊒⷗ߪߢ߈ߥ߆ߞߚ߇, Aဳߦߟ޿ߡߪBဳ ߣห᭽ߦ, ⅣႺਛߩᓸ↢‛ߦࠃࠆ᛫ේಽ⸃ᵴᕈ᷹ቯ߇น⢻ߣߥߞߚ. 3-1-4 ࿯ფᓸ↢‛ߦࠃࠆABOᑼⴊᶧဳߩᡷᄌ ᧄታ㛎ߩ⋡⊛ߪ, ⴊᶧဳ㐓ቯߩ⺋್ቯߩේ࿃ߣߥࠆᓸ↢‛ߩ⸃ᨆࠍ⋡⊛ߣߒߡ޿ߚ ߇, ABOᑼⴊᶧဳ᛫ේߩಽ⸃ᩣߩត⚝ߪ, ߘߩઁߦ߽ߐ߹ߑ߹ߥℂ↱߆ࠄ⎇ⓥߐࠇߡ޿ ࠆ. ㄭᐕ, ャⴊߦ↪޿ࠆⴊᶧࠍή㚝ߦߒߥ޿ߚ߼ߦ, Aဳ, Bဳ޽ࠆ޿ߪABဳߩ⿒ⴊ⃿ߩ