以高效能分子篩層析法與酵素連結免疫分析法分析西洋參水溶性不可消化多醣之分子結構特徵

୯ҥᆵ᡼εᏢғނၗྍᄤၭᏢଣ१ࠔࣽמࣴز܌

ᅺγፕЎ

Graduate Institute of Food Science and Technology College of Bioresources and Agriculture

National Taiwan University Master Thesis

аଯਏૈϩηᑔቫ݋ݤᆶሇનೱ่խࣝϩ݋ݤ ϩ݋ՋࢩୖНྋ܄όё੃ϯӭᗐϐϩη่ᄬ੝ቻ

Fingerprinting water-soluble nondigestible polysaccharides of Panax quinquefolius by high performance size exclusion

chromatography combined with enzyme-linked immunosorbent assay.

Ц၃൛

Shih-Ting, Wang

ࡰᏤ௲௤Ǻֈ׊ዞ റγ Advisor: Ting-Jang Lu, Ph. D.

ύ๮҇୯ 102 ԃ 7 Д

July, 2013

ᖴ ᖴᇞ

! ! གᖴৱৣ ֈ׊ዞറγӧ೭ٿԃύǴܭჴᡍǵፐ཰Ϸғࢲ΢ޑࡰᏤᆶᜢЈǴ٬

ᏢғૈӵයֹԋፕЎǴӧ೭ϐύᏢಞډԴৣჴ٣؃ࢂޑᄊࡋϷλЈᙣ཈ޑᆒઓǴჹ Դৣޑགᖴ੿ޑόࢂΟقٿᇟёа׎৒ޑǶ

! ! ፕЎ߃ዺ܍ᆾ ύ๮ዼᜪ१ࠔπ཰ࣴز܌܌ߏ ᐽ૽ԴৣǵᓉەεᏢ१ࠔᔼᎦ Ꮲس ஭҉کԴৣǵьϏ░Ԗज़Ϧљ୺Չߏ ᒘስᑢ ԴৣϷەើεᏢ१ࠔࣽᏢس ᓛᒴૈԴৣჹҁፕЎޑቩࢗǴඁᆶ஑཰ޑཀـᆶࡰ҅Ǵ٬ҁፕЎ׳ᑪֹऍǴӧԜు

߄གᐟǶ

! ! ٿԃޑᅺγғࢲ๊ჹόࢂҗԾρёаᐱԾֹԋǴሡाགᖴߚதӭΓޑᔅշǶག ᖴ ᖴలृԴৣǴӧྗഢࣴز܌Ե၂ය໔Ǵှเፐ཰΢ޑୢᚒǴаϷӧჴᡍ΢๏ϒ ޑၗྍڐշǴനख़ाޑࢂᖴԴৣ๏ךޑႴᓰǴ΋ޔࢂך߻຾ޑ୏ΚǶჴᡍ࠻ޑεৎǴ ᖴᖴգॺޑх৒Ǵᖴᖴដ୉ᏢۆߚதԖऐЈޑ௲ךჴᡍǴှ،ჴᡍ֚ᜤǴӵ݀ؒԖ یǴፕЎ΋ۓό཮೭ሶ໩ճǹᖴᖴဂඁᏢۆǴӧیي΢ᏢډΑ໒ਟ኷ᢀޑғࢲᄊࡋǴ ߥ࡭೭ኬޑߞۺǴ٣௃൩཮߆έԶှǹᖴᖴഓ൛ᏢۆǴӧჴᡍ΢ޑࡰᏤϷ࿶ᡍޑϩ ٦Ǵی଺٣ޑᇡ੿ᆶ୺๱ߚதзΓ൧ལǹᖴᖴ᜽ባᏢۆϷຽࢋᏢۆǴԖیॺޑࡰᏤ Ϸᔅշ٬ჴᡍ໩ճளӭǹᖴᖴऎ఼Ꮲۆᔅך຾ՉಒझჴᡍǴᗨฅࡕٰኧᏵؒԖҔ΢Ǵ ᗋࢂߚதགᖴی܌޸ޑਔ໔ᆶЈΚǹᖴᖴഩฐᏢۆǴیᇡ੿ޑᄊࡋ੿ޑࡐзΓལٵǴ གᖴی஥ሦך଺ჴᡍǴ੿ޑࢂڙ੻ؼӭǹᖴᖴמӼᏢߏǴգଓ؃ფགྷޑᆒઓࡐॶள ךॺᏢಞǹᖴᖴႳϡᏢߏǴόӭ၉ޑգᕴࢂᓨᓨࣁჴᡍ࠻଺٣ǴჹፕЎޑा؃Ϸո ΚǴΨࢂךॺԖҞӅ࿏ޑǹᖴᖴ΋ଆѺܣޑუՔ⍌৥ᆶз݇Ǵό཮᚛ဌ፯ًޑך೿

ाམգॺޑߡًѐ΢ፐǴவᅺ΋অፐډᅺΒ଺ჴᡍޑВηǴԖգॺޑഉՔ೿ᡣ೭٤ ਔӀόېൂǹᖴᖴ޼๮Ǵ؂ԛၟیಠϺ೿ளډࡐεޑ҅य़ૈໆǹᖴᖴࢅےǴЎਜλ ЦηޑЎਜמೌჴӧࢂӭளзΓᡋᡦǴߚதᖴᖴգޑᔅԆǹᖴᖴਁӹӧ GC-MS ௲ ػ૽ግޑбрǴჹܭ٣௃ޑոΚᄊࡋ཮ᡣգԏᛘࡐӭޑǹᖴᖴᜩཁǴԖیӧǴऍ१ ൩ό໔ᘐǴགᖴی஥๏ჴᡍ࠻៿኷Ϸ᏾౛஦୍ޑٌधǹᖴᖴϘ࣓ᔅך࣮मЎᄔाа

Ϸࣁךॺ஥ٰ๷ࡓᇯޑऍ१ᆶЎϯǶନԜϐѦǴགᖴԢৎᆶጯৎॷрჴᡍᏔ׷Ǵ٬

ჴᡍᏹբ׳БߡǶᖴᖴ׵λۆǵڬλۆǵഋλۆϷڬӃғჹܭ१ࣽ܌ՉࡹϷᕴ୍٣

୍ޑбрᆶٌधǴӭᖝԖգॺω٬ள܌΢ޑၮբֹ᏾Ƕ

! ! ᖴᖴܻ϶ॺӧЈᡫᆶᆒઓ΢䶒ךޑႴᓰǴᖴᖴ Oscar Lin ΋ޔӧךيᜐǴᖴᖴ G.j.Chen ࢂ҉ᇻޑคᆶউКޑऍ᜽Ǵᖴᖴ׵ۏଈǵጰோҦǵڬί՘Ǵգॺഉךوၸ ೭΋ࢤǴᖴᖴછ࿌ଈޑऍӳǴᖴᖴ஭ඵӹǵ໳ߪ◖کڑۗ㚎Ǵգॺ೿ࢂךΓғύࡐ ख़ाޑܻ϶Ƕ

! ! ᙣаԜፕЎ᝘๏നᒃངᆶЍ࡭ךޑৎΓǴᖴᖴݿݿǵ༰༰ǵۂۂჹךޑᜢЈǴ գॺޑᎦػϐৱаϷ຤Јਭ୻೷൩౜ӧޑךǹᖴᖴᚲނऔऔǵԺΤǵ໳ᓉॣᆶλ໳Ǵ ёངޑգॺ཮ᡣྠඊ੃ѨόـǴനࡕǴᖴᖴ΋ޔ೿ؒԖܫకޑԾρǶ

၃൛ ᙣᇞ ୯ҥѠ᡼εᏢ १ࠔࣽמࣴز܌

ύ๮҇୯ 102 ԃ 7 Д

ᄔ ᄔा

! ! ൂਲ਼לᡏςදၹ٬Ҕܭࣴز෌ނಒझ่ᄬǶҁፕЎᔕаሇનНှǵ഍ᚆηᐋિ

ቫ݋ǵଯਏૈϩηᑔቫ݋ݤ (high performance size excluion chromatography, HPSEC) ᆶሇનೱ่խࣝϩ݋ݤ (enzyme-linked immunosorbent assay, ELISA) ϩ݋Ջࢩୖ

Нྋ܄όё੃ϯӭᗐϐϩη่ᄬ੝ቻǶஒѐନೈқ፦ޑՋࢩୖಉӭᗐǴаሇનНှ

ନѐᐘણǴளډНྋ܄όё੃ϯӭᗐǴа഍ᚆηҬඤᐋિቫ݋س಍Ǵ٩ࢬࢱᡶᐚࡋ ϩᚆࣁ F1ǵF2ǵF3ǵϷ F4 Ѥঁ୔ϩǶஒӚ୔ϩа HPSEC ϩ݋Ǵམଛ ELISA assay

຾Չϩη่ᄬזೲᒣ᛽ᆶϯᏢϩ݋ዴᇡǴ٬Ҕ LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵ LM19ǵLM20 ᆶ JIM7 ຾ՉխࣝᒃکϸᔈǶځύ LM2 Ϸ LM5 ೏ᇡࣁёж߄ arabinogalactan type II (AGII) ᆶ arabinogalactan type I (AGI) ่ᄬǶF1 ᆶ F2 ӭᗐ

୔ϩܭᡶᐚࡋࣁ 0–0.1M ਔр౜Ǵಔԋࣁ arabinose ᆶ galactoseǴᆶ LM5ǵLM6 Ԗ ᒃکϸᔈǴ߄ҢڀԖ ß-(1→4)-galactan Ϸ linear arabinan ϩЍǶF3ǵF4 ӭᗐ୔ϩܭ ᡶᐚࡋࣁ 0.1–018M р౜ǴڀԖለ܄ᑗ galacturonic acid ᆶ glucuronic acidǴԜΒ୔

ϩନΑჹܭ LM2ǵLM5 Ϸ LM6 ԖᒃکϸᔈѦǴаለ܄ᑗࣁЬाಔԋϐ F4 ჹܭ LM19 ڀԖ࣬྽ଯޑᒃک܄ǴᡉҢԜΒ୔ϩڀԖ partially methyl esterified HG аϷ RGI ่ᄬᆶ AGIǵAGII Ϸ linear arabinan ฻ϩЍǴԜѦѤঁ୔ϩჹܭ LM10 ࣣคᒃ کϸᔈǴ٠όڀԖ xylogalacturonan ܈ xylan ฻่ᄬǶ

ᜢᗖӷǺଯਏૈϩηᑔቫ݋ݤǵሇનೱ่խࣝϩ݋ݤǵଯᏊໆ㸃ރਏᔈǵൂਲ਼ל ᡏǶ

Abstract

ġ ġ ġ This study used high performance size exclusion chromatography (HPSEC) and enzyme-linked immunosorbent assay (ELISA) to characterize the distribution of different epitopes within four fractions separated by DEAE chromatography of water soluble nondigestible polysaccharides. Water-soluble nondigestible polysaccharides were obtained from deproteinized crude polysaccharides subjected to starch hydrolyzing enzymes to remove the starch. DEAE chromatography can divide water-soluble nondigestible polysaccharides to four fractions (F1-F4) by different concentration of the eluent. Monoclonal antibodies have been widely used in studies of plant cell structures.

The mAbs LM2, LM5, LM6, LM7, LM10, LM19, LM20 and JIM7 were used in this research. The LM2 and LM5 resemble the AGII and AGI structures. F1 and F2 were observed when the eluent salt concentration was 0 to 0.1M. They are composed of arabinose and galactose, and the Mw are 71 and 116 kDa, respectively. Binding of F1 and F2 to LM5 and LM6 indicated the presence of RGI material with both arabinan and the AGI side chains. On the otherhand, F3 and F4 were observed when the eluent salt concentration was 0.1 to 0.18M. In addition to the arabinose and galactose, there are acidic polysaccharides such as galacturonic acid and glucuronic acid for F3 and F4.

Affinity to AGII (LM2), AGI (LM5), arabinan (LM6) and partially methyl esterified HG (LM19) were mainly observed in F3 and F4. It was also observed that F4 had a very high affinity to partially methyl esterified HG (LM19), indicating that it is mainly composed of an acid glycoprotein. Lastly, the four fractions had no observed immunoaffinity to LM10, indicating that there were no xylan or xylogalacturonan structures in the fractions.

Key word: high performance size excluion chromatography (HPSEC), enzyme-linked

Ҟ Ҟᒵ

ᖴᇞ ……….i

ᄔा ………...iii

Abstract ………...iv

Ҟᒵ ………I კҞᒵ ………...V ߄Ҟᒵ ………...VII ൘ǵ߻ق ... 1

ມǵЎ᝘ӣ៝ ... 2

ಃ΋കǵՋࢩୖ………2

ಃ΋࿯ǵғނϩᜪ ... 2

ಃΒ࿯ǵ׎ᄊ੝ቻ ... 2

ಃΟ࿯ǵࢲ܄ԋϩ ... 2

3.1 ӭᗐᜪ ... 3

3.2 جҒᜪ ... 3

ಃѤ࿯ǵғ౛ࢲ܄ ... 5

4.1 ׭ڋ࡚܄ڥ֎ၰੰੱ ... 5

4.2 ׭ڋဍዦғߏ ... 6

ಃΒകǵ݀ጤӭᗐ………7

ಃ΋࿯ǵ่ᄬᆶϩᜪ ... 7

1.1 Homogalacturonan (HGA) ... 8

1.2 Xylogalacturonan (XGA) ... 9

1.3 Rhamnogalacturonan-I (RG-I)………10

1.3.1 Arabinan………..10

1.3.2 Arabinogalactan type I (AGI)………..10

1.3.3 Arabinogalactan type II (AGII)………...11

1.4 Rhamnogalacturonan II (RG-II)………..11

ಃΒ࿯ǵ݀ጤӭᗐϐ่ᄬᆶғ౛ࢲ܄………...12

ಃΟകǵӭᗐޑϩᚆᆶપϯ………...15

ಃ΋࿯ǵϩભ؈ᐘݤ………...15

ಃΒ࿯ǵᡶ݋ݤ………...15

ಃΟ࿯ǵߎឦᒱӝݤ………...15

ಃѤ࿯ǵᆅࢊቫ݋ݤ………...16

4.1 ᚆηҬඤቫ݋ݤ……….16

4.1.1. ڰۓ࣬……….17

4.1.2. ࢬ୏࣬……….17

4.2 ϩηᑔᒧቫ݋ݤ……….17

4.2.1. ڰۓ࣬……….18

4.2.2. ౽୏࣬……….19

4.2.2.1.ׯᡂ pH ॶ………19

4.2.2.2.ׯᡂᡶᐚࡋ………...19

ಃѤകǵխࣝᔠෳБݤ………..……….20

ಃ΋࿯ǵ୷ᘵϟಏ………...20

ಃΒ࿯ǵלচᆶъלচᅿᜪ………...20

ಃΟ࿯ǵלᡏᅿᜪ………...21

ಃѤ࿯ǵሇનೱ่խࣝ֎ߕ၂ᡍ………...22

4.1 ڰۓ࣬……….22

4.2 ߔ༞……….22

4.3 ևՅᏊ……….22

4.4 ELISA БԄ……….23

ಃϖ࿯ǵൂਲ਼לᡏ………...25

5.1 LM2 ൂਲ਼לᡏ………25

5.2 LM5 ൂਲ਼לᡏ………25

5.3 LM6 ൂਲ਼לᡏ………26

5.4 LM7 ൂਲ਼לᡏ………26

5.5 LM10 ൂਲ਼לᡏ………..26

5.6 LM19 ൂਲ਼לᡏ………..27

5.7 LM20 ൂਲ਼לᡏ………..27

5.8 JIM7 ൂਲ਼לᡏ………...27

5.9 ൂਲ਼לᡏᆶ෌ނಒझᏛӭᗐᒃکϸᔈϐᔈҔ ... 28

ୖǵ၂ᡍࢬำკ ... 32

စǵ׷਑ᆶБݤ ... 33

ಃ΋കǵჴᡍ׷਑………..33

ಃ΋࿯ǵՋࢩୖኬࠔ ... 33

1.1 ٰྍ ... 33

1.2 ዗Н๧ڗނϐᇙഢ ... 33

1.3 Нྋ܄ಉӭᗐϐᇙഢ ... 33

1.4 Нྋ܄ё੃ϯӭᗐϷόё੃ϯӭᗐϐᇙഢ ... 33

1.5 ഍ᚆηҬඤᐋિቫ݋ϐНྋ܄όё੃ϯӭᗐ୔ϩ ... 34

ಃΒ࿯ǵჴᡍᛰࠔᆶ၂Ꮚ ... 35

2.1 ϯᏢᛰࠔᆶ၂Ꮚ ... 35

2.2 ኱ྗࠔ ... 36

2.3 לᡏ ... 36

2.4 ሇન ... 36

2.5.1.΋૓ᕗለ጗ፂనଛᇙ ... 36

2.5.2.όӕ pH ॶϐᕗለ጗ፂనଛᇙ ... 36

2.5.3.όӕ NaCl ᐚࡋϐᕗለ጗ፂనଛᇙ... 37

ಃΟ࿯ǵሺᏔ೛ഢ ... 37

ಃѤ࿯ǵϯᏢϩ݋Бݤ ... 38

4.1 ᕴᗐ֖ໆෳۓ ... 38

4.2 α-D-glucose ֖ໆෳۓ ... 38

4.3 ⾺ᑗለ֖ໆෳۓ ... 38

4.4 ೈқ፦֖ໆෳۓ ... 39

4.5 KDO ֖ໆෳۓ ... 39

4.6 ሇનೱ่խࣝ֎ߕݤ ... 39

4.7 ൂᗐಔԋϩ݋ ... 40

4.7.1. ࡑෳኬࠔ߻ೀ౛………40

4.7.2. ΟࢧᎉለНှ………..………..40

4.7.3. Ҙ✊ϯϸᔈ………41

4.7.4. ϩ݋س಍………41

4.8 ϩηໆෳۓ ... 42

4.8.1. ࡑෳኬࠔ߻ೀ౛………42

4.8.2. ኱ྗࠔ………42

4.8.3. ϩ݋س಍………42

4.9 HPSEC Սᖄ ELISA assay ... 43

ಃϖ࿯ǵ಍ीϩ݋ ... 43

Ҵǵ่݀ᆶ૸ፕ ... 44

ಃ΋കǵНྋ܄όё੃ϯӭᗐ………..44

ಃ΋࿯ǵНྋ܄όё੃ϯӭᗐ୔ϩϐಔԋϩ݋ ... 44

ಃΒ࿯ǵНྋ܄όё੃ϯӭᗐύӚӭᗐ୔ϩϐϩηໆ ... 46

ಃΒകǵሇનೱ่խࣝ֎ߕϩ݋ݤന፾ϯచҹϩ݋………..50

ಃ΋࿯ǵCoating buffer ϐ pH ॶᆶᚆηமࡋჹלচ֎ߕϸᔈϐቹៜ .. 50

ಃΒ࿯ǵଯᏊໆ㸃ރਏᔈ ... 54

ಃΟകǵНྋ܄όё੃ϯӭᗐύӚӭᗐ୔ϩϐൂਲ਼לᡏᒃکΚ่݀……..61

ഌǵ่ፕ ... 69

ࢠǵୖԵЎ᝘ ... 71

਎ǵߕᒵ ... 87

კ

კҞᒵ

კ΋ǵՋࢩୖύجҒޑϯᏢ่ᄬǶ ... 5

კΒǵ݀ጤӭᗐ่ᄬǴЬाёϩࣁ Homogalacturonan (HGA)ǵ Rhamnogalacturonan-I (RG-I) Ϸ Rhamnogalacturonan-II (RG-II)Ƕ ... 8

კΟǵHomogalacturonan ่ᄬკǶ... 9

კѤǵXylogalacturonan ่ᄬკǶ... 9

კϖǵRhamnogalacturonan-I ่ᄬკǶ ... 10

კϤǵArabinan ่ᄬკǶ ... 10

კΎǵArabinogalactan type I ่ᄬკǶ ...11

კΖǵArabinogalactan type II ่ᄬკǶ ...11

კΐǵRhamnogalacturonan II ่ᄬკǶ ... 12

კΜǵ܌Ԗᡏᑈ (Vt)ǵᆅࢊᗭಈѦޑᡏᑈ (Vo)ǵᆅࢊᗭಈϣޑᡏᑈ (Vi) ᆶᅉ੮ ᡏᑈ(Ve)ϐᜢ߯ҢཀკǶ ... 18

კΜ΋ǵޔௗϷ໔ௗխࣝ֎ߕ၂ᡍǶ ... 23

კΜΒǵਂਆϷᝡݾխࣝ֎ߕ၂ᡍǶ ... 24

კΜΟǵൂਲ਼לᡏᒣᇡלচ،ۓՏ࿼ (epitope) ௢ෳӭᗐ่ᄬǶ... 29

კΜѤǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩ݋კǶ ... 46

კΜϖǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F1 ϩηໆკ᛼Ƕ ... 47

კΜϤǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F2 ϩηໆკ᛼Ƕ ... 48

კΜΎǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F3 ϩηໆკ᛼Ƕ ... 48

კΜΖǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F4 ϩηໆკ᛼Ƕ ... 49

კΜΐǵόӕᐚࡋ gum arabic ྋܭΒԛᇃᚖНᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ݋Ƕ ... 52

კΒΜǵόӕᐚࡋ gum arabic ྋܭᕗለ጗ፂనᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ݋Ƕ ... 52

კΒΜ΋ǵόӕᐚࡋ gum arabic ྋܭ HPSEC ࢬࢱనᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ ݋Ƕ ... 53

კΒΜΒǵόӕ pH ॶϐᕗለ጗ፂనჹܭ gum arabicǵcitrus pecitnǵ(1→5)-α-L- arabinan ᆶ 4-O-methyl-glucuronoxylan Ϸൂਲ਼לᡏ LM2ǵLM5ǵLM6 ᆶ LM10 ϐ֎ӀॶቹៜǶ ... 53

კΒΜΟǵόӕෛϯ໊ᐚࡋϐᕗለ጗ፂనჹܭ gum arabicǵcitrus pecitnǵ(1→5)- α-L-arabinan ᆶ 4-O-methyl-glucuronoxylan Ϸჹᔈϐൂਲ਼לᡏ LM2ǵLM5ǵ LM6 ᆶ LM10 ϐ֎ӀॶቹៜǶ ... 54

კΒΜѤǵόӕᐚࡋ኱ྗࠔྋܭᕗለ጗ፂనᆶൂਲ਼לᡏᒃکΚ่݀Ƕ ... 55

კΒΜϖǵόӕᐚࡋ኱ྗࠔྋܭ HPSEC ࢬࢱనᆶൂਲ਼לᡏᒃکΚ่݀Ƕ... 56 კΒΜϤǵόӕᐚࡋ gum arabic ྋܭᕗለ጗ፂనᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ

კΒΜΎǵόӕᐚࡋ gum arabic ྋܭ HPSEC ࢬࢱనᆶൂਲ਼לᡏ LM2 ϐᒃکΚ ϩ݋Ƕ ... 57 კΒΜΖǵόӕᐚࡋ citrus pectin ྋܭᕗለ጗ፂనᆶൂਲ਼לᡏ LM5 ϐᒃکΚϩ

݋Ƕ ... 57 კΒΜΐǵόӕᐚࡋ citrus pectin ྋܭ HPSEC ࢬࢱనᆶൂਲ਼לᡏ LM5 ϐᒃکΚ

ϩ݋Ƕ ... 58 კΟΜǵόӕᐚࡋ(1→5)-α-L-arabinan ྋܭᕗለ጗ፂనᆶൂਲ਼לᡏ LM6 ϐᒃکΚ

ϩ݋Ƕ ... 58 კΟΜ΋ǵόӕᐚࡋ(1→5)-α-L-arabinan ྋܭ HPSEC ࢬࢱనᆶൂਲ਼לᡏ LM6 ϐ

ᒃکΚϩ݋Ƕ ... 59 კΟΜΒǵόӕᐚࡋ 4-O-methyl-glucuronoxylan ྋܭᕗለ጗ፂనᆶൂਲ਼לᡏ

LM10 ϐᒃکΚϩ݋Ƕ ... 59 კΟΜΟǵόӕᐚࡋ 4-O-methyl-glucuronoxylan ྋܭ HPSEC ࢬࢱనᆶൂਲ਼לᡏ

LM10 ϐᒃکΚϩ݋Ƕ ... 60 კΟΜѤǵόӕᐚࡋ citrus pectin ྋܭᕗለ጗ፂనᆶൂਲ਼לᡏ LM20 ϐᒃکΚϩ

݋Ƕ ... 60 კΟΜϖǵόӕᐚࡋ citrus pectin ྋܭ HPSEC ࢬࢱనᆶൂਲ਼לᡏ LM20 ϐᒃکΚ

ϩ݋Ƕ ... 61 კΟΜϤǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F1 ᆶൂਲ਼לᡏ

LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ϐᒃکΚϩ݋Ƕ ... 64 კΟΜΎǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F2 ᆶൂਲ਼לᡏ

LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ϐᒃکΚϩ݋Ƕ ... 65 კΟΜΖǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F3 ᆶൂਲ਼לᡏ

LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ϐᒃکΚϩ݋Ƕ ... 66 კΟΜΐǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F4 ᆶൂਲ਼לᡏ

LM2ǵLM5ǵLM6ǵǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ϐᒃکΚϩ݋Ƕ ... 67

߄

߄Ҟᒵ

߄΋ǵНྋ܄όё੃ϯӭᗐ࿶җ DEAE ቫ݋܌ϩᚆޑѤᅿϩቫӭᗐϐ୷ҁಔԋǶ ... 45 ߄ΒǵНྋ܄όё੃ϯӭᗐ࿶җ DEAE ቫ݋܌ϩᚆϐѤঁ୔ϩӭᗐځӚձൂᗐಔ

ԋǶ ... 45 ߄ΟǵНྋ܄όё੃ϯӭᗐ࿶җ DEAE ቫ݋܌ϩᚆϐѤᅿ୔ϩӭᗐځӚձϩη

ໆǶ ... 49 ߄ѤǵНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐѤঁ୔ϩڀԖϐ݀ጤ่ᄬ΋ំ

߄Ƕ ... 68

൘

൘ǵġ߻ق

ġ ġ Ջࢩୖӭᗐឦܭ݀ጤӭᗐǴ೏ᇡࣁЬाගٮխࣝፓ࿯ࢲ܄Ǵӵڈᐟ TNF-αǵ IFN-γ Ϸ IL-2 ញܫǴёफ़եࢬՉ܄གߵᐒ౗ၲ 89%ǴҞ߻ςԖᛰࠔ΢ѱǴԶ݀ጤӭ ᗐځ่ᄬፄᚇǴа۳ࣁှ݋ࢲ܄ӭᗐϐֹ᏾่ᄬǴӭ຾Չ़ғϯǴ٬Ҕ GC-MS Ϸ NMR ฻ሺᏔמೌǴલᗺࣁሡाપࡋଯޑኬࠔϷ઻຤εໆޑਔ໔Ƕ

ġ ġ ൂਲ਼לᡏҞ߻ςදၹ٬Ҕܭࣴز෌ނಒझ่ᄬǴᒣձ෌ނಒझว৖ၸำύǴځ ӭᗐޑׯᡂ௃׎Ƕ

ġ ġ ҁ ፕ Ў ٬ Ҕ ଯ ਏ ૈ ϩ η ᑔ ቫ ݋ ݤ (high performance size excluion chromatography, HPSEC) མଛሇનೱ่խࣝϩ݋ݤ (enzyme-linked immunosorbent assay, ELISA),Ǵ٬Ҕൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7Ǵځלচ،ۓՏ࿼ϩձࣁ ß-linked glucuronic acidǵß-(1→4)-galactanǵα-(1→5)- L-arabinanǵß-(1→4)-xylan Ϸ partially methyl α-(1→4)-GalAǴᙖҗלᡏᆶלচ،ۓ Տ࿼ϐᒃک੝܄Ǵࡌҥ΋ᔠෳѳѠǴଞჹՋࢩୖНёྋ܄Ӛᅿӭᗐϐϩη่ᄬ੝ቻ

຾Չϩ݋Ƕ

ມ

ມǵġЎ᝘ӣ៝

ಃ΋കǵġՋࢩୖ(Panax quinquefolius Linn)

ՋࢩୖǴΞӜ޸ᄡୖǵણӀୖǵՋࢩΓୖǵቶܿΓୖǵࢩୖ฻ǴচౢԾऍ ୯ч೽ډу৾εࠄ೽Ǵܭ࠶ථநٌԀౢໆࣁനεے(Wang and Yuan, 2008)ǶȨҁ૛ӆ

ཥȩᇡࣁՋࢩୖԖڰᆒӼઓϐਏǴȨᙴᏢ૱ύୖՋᒵȩ߾ᇡࣁځૈံշ਻ϩǴঋૈ

ံ੻ՈϩǴࣁځ܄థԶံǴΥటҔΓୖԶόڙΓୖϐྕံޣࣣёаԜжϐǶᛰ܄ࣁ ښҒǵधǵ܄థǶΕЈǵޤǵ๝Ο࿶ǴڀԖ੻ޤ഍ǵమ຀ОǵᎦगғࢭϐਏǴҔܭ ޤ຀Φࠏǵ຀዗ྠ॰฻ੱ(஭,2003)Ƕ

ಃ΋࿯ġǵғނϩᜪ

! ! Ջࢩୖࣁϖуࣽ (Araliaceae)ǵΓୖឦ (Panax)Ǵӭԃғ૛ҁ෌ނǶӕឦ෌ނԖ

Panax ginseng C. A. Meyer (٥ࢪΓୖ)ǵ Panax notoginseng (Burk.) F. H. Chen

ಃΒ࿯ġǵ׎ᄊ੝ቻ

! ! ෌ਲ਼ӄᡏคЛǴਥϩࣁЬਥǵ݄ਥکᠣਥǴԺ፦Ǵ߄य़Ԗుభಉಒό฻ޑᐉદǶ όӕԃសՋࢩୖЬਥᒿ⭂សቚуǴڬҜǵԛғ໧Ҝ೽کЕ፦೽य़ᑈǴ֡ևቚуᖿ༈Ƕ ਥޑᖓᏛಔᙃಒझϣᓯᙒᙦ൤ޑᐘણಈǴӧวػғߏύǴև౜΋ۓೕࡓ܄ޑᡂϯǶ ਥکӦ΢ಳҬ཮ೀԖ΋ӦΠಳǴᆀࣁਥಳǴዬރǴಳࣁ༝ࢊ׎ǶඓރϖрፄယǴယ ևॹռ׎܈ߏᐍ༝׎ǴӃᆄँӾǴᜐጔڀԖόೕ߾ಉᒯᏁǴ୷೽ևུ׎Ƕദࠠ޸ׇǴ ዀ݀ࡧ༝׎Ǵԋዕࣁుआ(೾,1990)Ƕ

ಃΟ࿯ġǵࢲ܄ԋϩ

! ! Γୖਥ଺ࣁᛰ׷ςຬၸٿίᎩԃǴߏΦаٰ೏ຎࣁᡫϏ֮ᛰ (panacea)Ǵ

ڀԖۯԃ੻ტϐфਏǴࢲ܄ނ፦ЬाࣁӭᗐᜪǵجҒᜪǵᴏ两ᜪǵᆫΌᬨᎇ (polyacetylenic alcohol) Ϸિެለ฻(Lee, 1992; Attele et al., 1999) Ƕ

3.1

! ! ՋࢩୖӭᗐڀԖቚ຾խࣝϸᔈޑૈΚǴڈᐟరЃಒझ (lymphocyte) ǵϟ қ፦ (interleukin) ǵဍዦᚯԝӢη (tumor necrosis factor) ࢲ܄(Ma et al., 1998;

Assinewe et al., 2002) Ƕ а Ջ ࢩ ୖ ӭ ᗐ ೀ ౛ λ Ⴕ ࡕ ག ࢉ қ Յ ۺ ੧ ๵ (Candida

albicans)Ǵೀ౛ಔ࣬ၨܭჹྣಔܴᡉफ़եқՅۺ੧๵ܭ๝᠌ޑਏሽ (titer)ǵ෧Ͽ๝

᠌ ᆶՈమ ύ วݹಒ झ Ӣη (inflammatory cytokine) MCP-1 ᆶ MIP-2 ޑ ౢғ (Trammell et al., 2012)Ǵ٠Ъӧᡏϣ (in vivo) ჴᡍύว౜ǴՋࢩୖӭᗐቚуߎ໳Յ ဟ๻ౚ๵ (Staphylococcus aureus) གࢉλႵϐӸࢲ౗ǵ෧ϿࢲϯѮᏘಒझϷՈనύ

༾ғނ֖ໆ(Lim et al., 2002; Ahn et al., 2006)ǶჹܭޤဍዦλႵ (Lewis tumor-bearing C57BL/6 mice)ǴՋࢩୖӭᗐᡉ๱׭ڋဍዦғߏǵቚуխࣝᏔ۔ϐૅဏϷ๠᠌ख़ໆǴ ڈᐟ IL-2 Ϸ IFN-γ ߄౜ǴҗԜёޕǴՋࢩୖӭᗐаࢲϯ Th-1 ಒझࣁЬ(Zhu et al., 2012)Ƕ

3.2

! ! جҒࣁՋࢩୖޑЬाࢲ܄ԋϩǴҞ߻ςԖӭᅿجҒᜪ೏ϩᚆǴϯᏢ่ᄬ

΢ᆶᎇᜪᗖ่ޑᑗᜪࣁဟ๻ᑗǵഝ޵ᑗǵ݀ᑗϷጧᑗ(Gillis, 1997; Yoshikawa et al., 1998)Ǵܭ C-3ǵC-6 ܈ C-20 Տ࿼ǴӢᑗಔԋϷᗖ่Տ࿼ৡ౦Ꮴठόӕޑғ౛ࢲ܄

(Attele et al., 1999)ǶRh1 ᆶ Rh2 ่ᄬ࣬՟Ǵόӕᗺࣁ ß-D-glucopyranosyl group ᗖ

่ܭ Rh1 ޑ C-6 ᆶ Rh2 ޑ C-3 Տ࿼ǴRh2 ёа׭ڋ B16-BL6 ໵Յનዦಒझғߏǵ ڈᐟ໵ՅનғӝԋϷಒझ໔ᗹߕ܄ǴԶ Rh1 ჹܭಒझࠅคԜբҔ(Odashima et al., 1985)ǶՋࢩୖجҒᜪύǴRb1 Ϸ Rg1 ڀԖڈᐟǵ׭ڋύኰઓ࿶س಍Ϸፓ࿯ઓ࿶໺

ᏤϐբҔǶύኰᖌᡵૈس಍ (Central cholinergic systems) ॄೢᏢಞϷ૶Ꮻၸำ (Perry, 1986)ǴRb1 ቚуس಍ύᖌᡵޑឪڗໆ(Benishin, 1992)Ǵ٬Όㄽᖌᡵҗੇଭ଑

ύញܫ(Benishin et al., 1991)Ǵ຾ԶளޕՋࢩୖجҒڀԖߦ຾Ꮲಞǵ૶ᏫၸำϷઓ࿶

ғߏૈΚ(Takemoto et al., 1984; Salim et al., 1997)ǶTsang ฻ΓࡰрǴՋࢩୖ๧ڗނ

׭ڋ GABA ǵك਽ለ (glutamate)ǵӭЃữ (dopamine) ǵ҅๝΢ဏન (noradrealin) Ϸ Ո మ ન (serotonin) ܭεႵတ ँ᝻ λᡏޑ ֎ԏڀԖ ᐚࡋ ਏᔈ (concentration- dependent) (Tsang et al., 1985a)ǶՋࢩୖ๧ڗނᆶ GABAAڙᡏ่ӝϐଛᡏ (ligand) ໔ޑբҔǴቹៜ GABAergic ઓ࿶໺ᏤǴࣁՋࢩୖϐख़ाࢲ܄ԋϩ(Yuan et al., 1998)Ƕ ନԜϐѦǴKenarova ว౜ǴRg1 ᙖҗߦ຾ᇶշࠠ T ಒझ (T helper cell)ǵT రЃಒ झ (T lymphocyte) ᆶԾฅఠЋಒझ (NK cell) ғԋǴගϲխࣝϸᔈ(Kenarova et al., 1990)Ƕ

კ΋ǵՋࢩୖύجҒޑϯᏢ่ᄬǶ

Figure 1. Structures of ginsenosides discussed in the text. Based on chemical structure, there are two major groups: panaxadiols (A) and panaxatriols (B). Rh3, as shown in the lower part of (A), differs from other panaxadiols at the side chain. Ginsenoside Ro, a nonsteroidal saponin, is shown in (C).(Attele et al., 1999)

ಃ

ಃѤ࿯ġǵғ౛ࢲ܄

4.1

! ! Cytotoxic T lymphocytes or CTLs ࢂҗ Tc (T cytotoxic) ಒझౢғǴڀԖᒣᇡǵ

੃ྐᎁڙׯᡂޑԾᡏಒझ (altered self cell)ǴӵੰࢥགࢉಒझǵဍዦಒझǶ΋૓Զ قǴCTLs ࣁ CD8⁺Ǵឦܭ class I MHCǶCTL ୖᆶޑխࣝϸᔈёϩࣁٿঁ໘ࢤǴ२ Ӄ native Tc ಒझୖᆶࢲϯϷϩϯࣁ functional effector CTLs ǹӆޣǴeffector CTLs ᒣᇡלচǴ٬ϐ஝ᚯǶNative Tc ಒझคݤ੃ྐלচǴ೏ຎࣁ CTL precursor (CTL-

P)Ǵ୤Ԗ྽ CTL-P ೏ࢲϯࡕωڀഢ੃ྐѦٰಒझૈΚǶӭኧಒझߞ৲ஒ CTL-P ࢲ ϯϷቚ෗Ǵځ΋ࣁ IL-2 (interleukin-2)ǶIL-2 ࣁख़ा T ಒझԋߏӢηǴቚуխࣝౚ

ೈқӝԋǵԾฅఠЋಒझޑಒझྋှૈΚ (cytolytic activity) (Leonard, 1999)Ƕӧ IL- 2 knock out λႵύว౜ǴલϿ IL-2 ߾ቲЗ CTL фૈǴ྽לচ೏మନϐࡕǴIL-2 ֖ ໆ෧ϿǴёᇨᏤ CTL ಒझ঒ΫวғǴ่״խࣝϸᔈ(Thomas et al., 2007)ǶCOLD- fX (CVT-E002) ࢂҗՋࢩୖਥ೽๧ڗϐံкࠔǴ൤֖ poly-furanosyl-pyranosyl- saccharidesǴӵႵ׵ᑗǵဟ๻ᑗǵъ٢ᑗǵߓ܎դᑗϷъ٢ᑗ⾺ለǶа COLD-fX ೀ

౛ڙ Con-A ᇨᏤλႵ๠᠌ಒझϷڙࢬՉ܄གߵੰࢥགࢉϐΓᜪڬᜐՈనൂਡౚಒ झ (peripheral blood mononuclear cell) Ǵࣣܴᡉቚу IL-2 Ϸ IFN-γ ౢғ(Wang et al., 2004; McElhaney et al., 2006)ǶMcElhaney ฻Γஒ COLD-fX ܭࢬՉ܄གߵӳวۑ࿯

๏ϒ 198 ՏԋΓܺҔၲ 4 ঁДǴफ़եӢࣁࢬགੰࢥ (influenza) ܈ڥ֎ၰᑼӝੰࢥ (respiratory syncytial virus) ܌Їวϐ࡚܄ڥ֎ၰੰੱ (acute respiratory illness, ARI)

ၲ 89%ǴԶࡕܕ໣ 43 ՏǴ65 ྃа΢ޑ଼நԋԃΓǴ22 Տ๏ϒ COLD-fXǴځᎩ 21 Տ๏ϒӼኃᏊǴ4 ຼࡕݙ৔ࢬՉ܄གߵࣝभǴ่݀ᡉҢǴܺҔ COLD-fX ಔձၨჹ

ྣಔ෧Ͽ 48% ᐒ౗ᑡ஻΢ڥ֎ၰགࢉ੯ੰ(McElhaney et al., 2004; McElhaney et al., 2006)Ƕ

4.2

! ! p21 (ಒझຼය׭ڋೈқ,cyclin inhibitor protein) ࣁלဍዦࢲ܄ϐ΋ख़ाᜢᗖ Ǵᗖ่ܭಒझຼය٩ᒘᐟ䁙 (cyclin-dependent kinase, CDK)Ǵ׭ڋሇનࢲ܄ǵߔЗ DNA ӝԋϷߔᘐಒझຼයǴ٠ԖࣴزᡉҢǴՋࢩୖ๧ڗނᙖҗϣӧ঒Ϋ೼৩Ǵׯ ᡂ BaxǵBidǵcaspase-3 ᆶ PARP ฻ೈқ፦߄౜ǴаϷಈጕᡏጢႝՏᆶ cytochrome c ញܫǴߦ຾ဍዦಒझԝΫ(Oh and Lee, 2004; Oh et al., 2005; Yang et al., 2006; Koo

et al., 2007)ǶаՋࢩୖ๧ڗނೀ౛ HCT116 Γᜪ่ဉᕎಒझ 48 λਔǴՋࢩୖ๧ڗ

౜ೀ౛ࡕಒझϐ p53ǵp21 Ϸߦಒझ঒Ϋೈқ Bax ֖ໆࣣቚуǴphospho-MEK ߾෧ ϿǴԖշܭߔᘐಒझຼයǵ෧Ͽಒझߞ৲(King and Murphy, 2010)Ƕ

ಃ

ಃΒകǵġ݀ጤӭᗐ

! ! ݀ጤࣁԾฅࣚύፄᚇޑӭᗐ่ᄬǴ35%Տܭᚈηယ෌ނϷߚңҁࣽൂηယ෌

ނϐ߃ભಒझᏛǴ2-10%ܭ૛ҁ෌ނǵ5%ܭЕҁ෌ނಔᙃ(O'Neill et al., 1990; Ridley

et al., 2001)ǴЬाӸӧ຾Չғߏϩ຋ϐಒझᏛϷύጤቫ฻Ƕ݀ጤᔅշ෌ނғߏǵว

et al., 2001)ǶᔈҔܭ१ࠔБय़Ǵ଺ࣁԋጤᏊϷᛙۓᏊǴςԖࣴزࡰрǴ݀ጤёफ़ե

Jackson et al., 2007)Ƕ

ಃ΋࿯ġǵ่ᄬᆶϩᜪ

! ! ݀ጤӭᗐҔа߄Ң൤֖ъ٢ᑗ⾺ለޑӭᗐǴԿϿ 10 ঁൂᡏ܌ಔԋϐߏ᜘ъ٢ ᗐ⾺ለ୔ (homogalacturonan)(Nothnagel et al., 1983; Samuelsen et al., 1996a)ǴΨத ـߓ܎դᑗǵъ٢ᑗǵႵ׵ᑗ฻ύ܄ᑗǶለ܄౦፦ӭᗐǴڀӭϩη܄ (polymolecular) Ϸӭϩණ܄ (polydisperse)ǴϩηໆϩѲጄൎଯၲ 100 kDaǴځಔԋ཮Ӣࣁ෌ނٰྍǵ

๧ڗБԄ฻Զ౦Ƕ݀ጤӭᗐёᘜયр΋ڂ่ࠠᄬǴମࢎࣁ 1ʈ4 ᗖ่ޑъ٢ᑗ⾺ለǴ Ь ा ϩ ࣁ Homogalacturonan (HGA) ǵ Rhamnogalacturonan-I (RG-I) Ϸ Rhamnogalacturonan-II (RG-II) (Pérez S et al., 2003)Ƕ

კΒǵ݀ጤӭᗐ่ᄬǴЬाёϩࣁ Homogalacturonan (HGA)ǵ Rhamnogalacturonan-I (RG-I) Ϸ Rhamnogalacturonan-II (RG-II)Ƕ

Figure 2. Schematic representation of the “canonical” primary structure of pectins. For the sake of simplicity, the schematic representation of HGA, RG-I, and RG-II is given assuming that these three domains are covalently linked, although this points is not firmly established(Pérez S et al., 2003).

1.1 Homogalacturonan (HGA)

! ! ̄omogalacturonan ࣁ 1,4 ᗖ่ޑޔ᜘ъ٢ᑗ⾺ለ (GalpA) ่ᄬǴ՞݀ጤ 65%Ǵ

೽ϩ♐୷΢ԖҘ୷অႬǴܭ C-3 ܈ C-2 Տ࿼ڀΌㄽ୷(Ishii, 1995; 1997)ǴЍ᜘ၨϿǴ Ξᆀࣁѳྖ୔ (smooth region)Ƕ҂Ҙ୷ϯޑ HGA ஥ॄႝǴᆶ Ca²⁺բҔ׎ԋᛙۓጤ ᡏǴࢂ݀ጤϐЬाᏉጤᐒڋ(Liners et al., 1989; Jarvis and Apperley, 1995)Ƕ

კΟǵHomogalacturonan ่ᄬკǶ

Figure 3. The primary structure of homogalacturonan(Ridley et al., 2001)

1.2 Xylogalacturonan (XGA)

! ! Xylogalacturonan ࣁ homogalacturonan ځ C-3 Տ࿼а ß-D-xylose ڗж(Aspinall, 1980; O'Neill et al., 1990; Schols et al., 1995; Kikuchi et al., 1996; Yu and Mort, 1996)Ǵ

໻Ӹܭғ෗ಔᙃǴӵلᜪᅿηǵ݀ჴϷ޸ણǶࣴزว౜Ǵӝԋ xylogalacturonan ࢂ Ҕаჹלੰচ๵װᔐǴ٬ homogalacturonan ၨૈܢלੰচ๵ౢғϐϣᆫъ٢ᑗ䁙 (endopolygalacturonase) (Jensen et al., 2008)Ƕ

კѤǵXylogalacturonan ่ᄬკǶ

Figure 4. Structure of a part of xylogalacturonan(O'Neill et al., 1990)

1.3 Rhamnogalacturonan-I (RG-I)

! ! Rhamnogalacturonan ޑ่ᄬЬाࣁ→4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→Ǵ՞݀

ጤ 20-35%ǴGalpA ޑ C-3 ܈ C-2 Տ࿼Όㄽϯ(Komalavilas and Mort, 1989)ǴRhap ޑ C-4 Տ࿼߾೏ύ܄Ϸለ܄ᑗڗжǴ܈ೱௗ α-1,5 ᗖ่ޑߓ܎դᑗ (α-1,5-linked-L-Araf) Ϸ ß-1,4 ᗖ่ޑъ٢ᑗ (ß-1,4-linked-D-Galp) ׎ԋୁЍ(O'Neill et al., 1990; Carpita and Gibeaut, 1993)Ǵёϩࣁ ArabinanǵArabinogalactan type I Ϸ Arabinogalactan type IIǶԜ୔ၨӭϩЍǴΞᆀࣁӭЛ୔ (hairy region, or ramified region) (Nakamura et al., 2002; Paulsen and Barsett, 2005)Ƕ

კϖǵRhamnogalacturonan-I ่ᄬკǶ

Figure 5. Structure of a part of rhamnogalacturonan-I(Komalavilas and Mort, 1989)

1.3.1 Arabinan

! ! Arabinan җ L-arabinose ܌ಔԋǴதௗܭ݀ጤ galactan ΢ǴЬाࣁ 3,5 ᗖ่ǴӢ

ٰྍόӕԶԖޔ᜘܈ୁЍ่ᄬǶ΋૓ԶقǴମ༸а 1ʈ5 linking ࣁЬǴϩЍЬाӧ C-3 Տ࿼ǴC-2 ࣁୋǶ๧ڗၸำ٬Ҕሇન܈ለНှёஒځញܫрٰ(Paulsen and Barsett, 2005)Ƕ

კϤǵArabinan ่ᄬკǶ

Figure 6. Structure of a part of an arabinan(Paulsen and Barsett, 2005).

1.3.2Arabinogalactan type I (AGI)

! ! Arabinogalactan type I ࣁ ß-1,4-linked-D-galactanǴӧ C-3 Տ࿼ௗа arabinan ᄬ

ԋϩЍ(Jermyn and Yeow, 1975; Van Holst and Clarke, 1986; Huisman et al., 2001)Ƕ

კΎǵArabinogalactan type I ่ᄬკǶ

Figure 7. Structure of Arabinogalactan type I (Clarke et al., 1979)

1.3.3 Arabinogalactan type II (AGII)

! ! Arabinogalactan type II ࣁ ß-(1ʈ3)-D-galactanǴЍ᜘ ß (1ʈ6)-D-galactan ߾த೏

Araf ܈ ArapǵRhamǵXylǵGlcA ܌ڗжǴϩЍᗺӧ 1,3,6-linking GalǴڀԖଯࡋϩ ЍǶAGII ᆶ Yariv reagent ౢғआՅ؈ᐘ(Jermyn and Yeow, 1975; Van Holst and Clarke, 1986)ǶAGII தᆶೈқ፦่ӝᄬԋ Arabinogalactan protein (AGP)ǴAGII ܌՞Кٯε ܭ 90%Ǵೈқ፦໻՞ϿໆǶ(Pellerin et al., 1995; Luonteri et al., 2003)

კΖǵArabinogalactan type II ่ᄬკǶ

Figure 8. Structure of Arabinogalactan type II(Redgwell et al., 2002).

1.4 Rhamnogalacturonan II (RG-II)

! ! RG-II ࣁಔԋፄᚇޑӭᗐǴ໻՞݀ጤϐ΋λ೽ϩǴऊ 10%(O'Neill et al., 2004)Ƕ

Ь༸җ 8 ঁа΢ϐъ٢ᑗ⾺ለа α-1,4 ᗖ่܌ᄬԋǴܭ C-3 ܈ C-4 Տ࿼ೱௗԿϿ 12 ᅿᑗ୷Ǵ׎ԋ 20 ᅿа΢ᗖ่Ǵཷౣёϩࣁϖᅿ (A~E)Ǵၨڀ੝ՅޑࢂϩЍύр౜

شـޑൂᗐࣁ 2-O-methylfucoseǵ2-O-methylxyloseǵapioseǵaceric acidǵ2-keto-3- deoxy-D-manno-octulosonic acid (KDO) Ϸ 3-deoxy-D-lyxo-2-heptulosaric acid (DHA) (O'Neill et al., 1990; Doco et al., 1997; Pérez S et al., 2003; O'Neill et al., 2004)Ƕځύ KDO Ϸ DHA Ь ा Ӹ ӧ ܭ ਱ ើ М ഍ ܄ ๵ (gram-negative bacterial) ޑ િ ӭ ᗐ (lipopolysaccharides) ύǴԶ aceric acid ߾ѝ೏ว౜ܭ RG-II ύǴӢԜ KDOǵDHA Ϸ aceric acid ё೏྽բ RG-II ϐ੝ቻ܄ൂᗐǴᙖа߃؁ղᘐ RG-II ϐӸӧ(York et

al., 1985)ǶಒझᏛ΢ऩ෧Ͽ RG-II Βᆫᡏ׎ԋǴ཮ᄬԋᝄख़ޑғߏલഐǴӵٹᏂੱǴ

ࡺ RG-II ޑΒᆫϯჹܭ෌ނғߏวػཱུࣁख़ा(Mohnen, 2008)Ƕ

კΐǵRhamnogalacturonan II ่ᄬკǶ

Figure 9. Schematic representation of the primary structure of RG-II monomer(Pérez S

et al., 2003)

ಃ

ಃΒ࿯ġǵ݀ጤӭᗐϐ่ᄬᆶғ౛ࢲ܄

! ! ύ૛ᛰ෌ނύޑ݀ጤӭኧڀԖלံᡏࢲ܄Ǵӵ྽ᘜ (Angelica

acutiloba)ǵਮच (Bupleurum falcatum) (Kiyohara et al., 1988; 1989a)ǵΓୖ (Panax ginseng) (Gao et al., 1988; Gao et al., 1990)ǵҒ૛ (Glycyrrhiza uralensis) (Zhao et al.,

et al., 1979)ǴҔܭᔠෳ (1→3,6)-ß-galactan ֖ໆ(Holst and Clarke, 1985; Kiyohara et al., 1989b; Gao et al., 1991)Ƕ

! ! ݀ጤޑለ܄ӭᗐϐύǴೱௗԿ rhamnogalacturonan ޑЍ᜘ӵ arabinogalactan type I and II Ϸ 6-linked galacto-oligosaccharides ࢂࢲϯံᡏ alternative Ϸ classical ೼ ৩ޑЬा่ᄬǶGalacturonan ΢ऩԖҘ୷✊ϯ (type I) ܈൤֖Ѝ᜘ (type II) ਔǴ߾

཮׭ڋ ramified region ޑғ౛ࢲ܄Ƕ

! ! Honda ฻Γࣴزว౜Ǵ᜘ߏຬၸ 20 DP ޑӭᗐӵ ß-D-(1→3)-glucan ᆶ

alternative pathway ޑࢲ܄࣬ᜢǴԶҘ୷ (methyl group) ཮׭ڋ ß-D-(1→3)-glucan ࢲ܄Ǵ຾΋؁ឍॊӭᗐޑҥᡏ่ᄬҭ཮ቹៜځ੝܄(Honda et al., 1986)Ƕ

! ! ΟᅿҗΓୖ (P. ginseng) ϩᚆрٰޑ݀ጤӭᗐ (GL-PIǵPIIǵPIV) ڀԖ לံᡏࢲ܄(Gao et al., 1988)ǴGL-PIII ߾คǴКၨӭᗐϐ໔ޑ่ᄬǴว౜ GL-PIII ڀԖၨӭڀϩЍޑ galacturonanǴᇥܴ galacturonan ΢ޑЍ᜘཮ቹៜځࢲ܄(Gao et

al., 1990)Ƕ

! ! AGIIa ࢂҗ྽ᘜ (A. acutiloba) ዗Н๧ڗనϩᚆԶளޑלံᡏӭᗐ (ځᎩϝԖ AGIIb-1 ǵ AR-2IIa ǵ AR-2IIb ǵ AR-2IIc Ϸ AR-2IId) Ǵ २ ԛ ว ౜ ύ ૛ ᛰ ύ ޑ

arabinogalactan ڀԖࢲϯံᡏфૈ(Yamada et al., 1985a)ǶAGIIa э྽ᘜӭᗐ໻΋λ

೽ϩǴࠅڀԖؼӳޑғ౛ࢲ܄ǶAGIIa ࣁ arabino ß-3,6-galactanǴڀԖ 5-linked α-L- arabinose Ϸ 3-linked ß-D-galactose ڗж୷Ǵ࿶җ alternative ᆶ classical ೼৩ࢲϯ

ံᡏǶࣴزว౜ǴӭኧڀԖࢲϯံᡏфૈޑ arabinogalactan ࣁ type IIǴՠࢂҗ larch wood ϩᚆрޑ type II arabinogalactan ߾όڀԖࢲϯံᡏϐфਏǶAR-2IIaǵ2IIbǵ 2IIc Ϸ 2IIdǴځ่ᄬࣁ 90%а΢ޑ galacturonan Ϸ ramified region(Kiyohara et al., 1988) Ƕ Kiyohara ฻ Γ а endo-α-(1→4) polygalacturonase Ϸ ೀ ౛ ಥ ✊ ϯ (de- esterification) ݀ጤǴᇙഢ ramified region ኬࠔǴว౜Ԝ୔ၨচۈ݀ጤڀԖࢲϯံᡏ ࢲ܄ǴЪ AR-2IIa ޑ ramified region ڀԖεໆ(1→3,6)-ß-galacto-oligosaccharide chainsǴ ᡉҢ ramified region ࢂҗ ß-3,6-galactan ܌ᄬԋ٠ຎࣁࢲϯံᡏޑࢲ܄୔ୱ(Kiyohara

et al., 1988; 1989a)ǶAGIIb-1 ύלံᡏࢲ܄ޑᗐ᜘ёᙖҗሇનϷϯᏢफ़ှ܌ளޕ

(Yamada et al., 1987a; Zhang et al., 1996) Ƕ а arabinofuranosidase Ϸ exo-ß-D- (1→3)-galactanase ೀ౛ AGIIb-1ǴНှ ß-(1→3)-galactan ମࢎ(Tsumuraya et al., 1990) Ϸ arabinogalactan side chainsǴౢғεໆޑ galactosyl oligosaccharide chainsǴՠࢂ

rhamnogalacturonan Ϸځୁ᜘Ǵό཮೏ galactanase НှǴϝ߄౜ᆶ AGIIb-1 ࣬ӕϐ

ံᡏࢲϯࢲ܄Ǵ߄Ң AGIIb-1 ޑࢲϯံᡏࢲ܄Ьाࣁᗐ᜘ޑମࢎǴԶߚЍ᜘ǶZhang

฻Γа Smith degradation Ϸ exo-ß-D-(1→3)-galactanaseǴࡰр AGIIb-1 ޑମࢎࣁᡉ

๱ࢲϯံᡏޑъ٢ჲᗐ᜘Ǵа 3- and 3,6-linked galactosyl residues ܌ಔԋǴҭ߄Ң ß-(1→3)-galactan ࣁ AGIIb-1 ޑЬा่ᄬ(Zhang et al., 1996)Ƕ

! ! ೚ӭלံᡏ݀ጤӭᗐࣣ֖Ԗ arabino-3,6-galactan chainsǴᇥܴԜ่ᄬҭ

ࣁࢲ܄ӭᗐ(Yamada et al., 1985a; Yamada et al., 1986; Kiyohara et al., 1987; Yamada

et al., 1987a; Yamada et al., 1987c)ǶAGIIb-1 ࿶ለНှள arabino-3,6-galactan (N-

౛ N-I ࡕϝڀԖלံᡏࢲ܄Ƕஒ N-I аጤᡏቫ݋ࡕளύϩηໆӭᗐǴࣁ GN-1A Ϸ GN-1BǴࣣڀԖံᡏࢲϯ੝܄ǴԜࢲ܄่ᄬࣁ 6-linked galactosyl residuesǴ߄Ң N- I ޑЍ᜘Ьाҗ galactose ܌ᄬԋǴԶ 6-linked-ß-D-(1→3)-galactan ࣁࢲϯံᡏޑЬ

ाಔԋ(Kiyohara et al., 1997)Ƕ

! ! ࣁ຾΋؁ᕕှࢲ܄ӭᗐࢲϯံᡏᐒڋǴלᡏᆶࢲ܄ӭᗐޑᒃکϸᔈࣁ΋ؼӳ ᔠෳלচ،ۓՏ࿼ (epitope) ޑБݤǶҗਮचϩᚆளӭᗐ bupleuran 2IIb Ϸ 2IIcǴα

ܺᗯ१λႵǴ٬Ҕ੝ۓלᡏᔠෳלဍዦӭᗐ ramified region ϐϩѲ௃׎(Sakurai et

al., 1996)Ǵ่݀ว౜ӧط᠌Ϸ୻Мඬ (Peyer's patches) ύǴԖ੝ۓϩηໆޑӭᗐᅉ

ಃ

ಃΟകǵġӭᗐޑϩᚆᆶપϯ

ಃ΋࿯ġǵϩભ؈ᐘݤ

! ! ϩભ؈ᐘݤࢂਥᏵόӕӭᗐӧόӕᐚࡋᎇǵ✉ύڀԖόӕྋှࡋޑ܄፦Ǵவλ ډεࡪКٯуΕҘᎇǵΌᎇ܈Ч✉຾Չϩભ؈ᐘ(ᇳ et al., 2007)Ƕ

ಃΒ࿯ġǵᡶ݋ݤ

! ! ਥᏵόӕӭᗐӧόӕᡶᐚࡋύྋှࡋόӕԶஒځϩᚆޑ΋ᅿБݤǴதҔޑᡶ

݋ྋనԖෛϯ໊ǵෛϯႇϷ౷ለሓ฻(ᇳ et al., 2007)Ƕ

ಃΟ࿯ġǵߎឦᒱӝݤ

! ! ճҔӭᗐૈᆶልǵ᎕ǵ້Ϸႉᚆη׎ԋᒱӝނԶ؈ᐘǴதҔᒱӝᏊԖත݅၂Ꮚǵ

ෛϯልǵణ਼ϯ᎕کᎉለႉ฻(ᇳ et al., 2007)Ƕ

ಃ

ಃѤ࿯ġǵᆅࢊቫ݋ݤ

4.1 ᚆηҬඤቫ݋ݤ (ion exchange chromatography)

! ! ᚆηҬඤቫ݋ݤࣁதҔϩᚆБݤǴڗ،ܭௗӧೈқ፦ (܈ځдϩ݋ނ) ΢ޑ҅

܈ॄႝ୷იǴڰۓܭڰۓ࣬Ǵӧ֎ߕၸำύǴ౽୏࣬೏ҬඤǶᚆηҬඤᐋિቫ݋ݤ ёϩࣁೱ่ (binding) کѐ֎ߕ (desorption) ϐၸำǴϩ݋ނೱ่ܭڰۓ࣬Ǵҗܭ ᡶᚆηᐚࡋ܈ pH ॶޑೱុ܈ఊࡋׯᡂǴ෧১ϩ݋ނᆶڰۓ࣬໔ޑҬϕբҔǴၲډ ϩᚆਏ݀Ƕ΋૓ԶقǴ֎ߕکှନ֎ߕޑၸำࢂҗΟ࣬ϐ࣬ϕբҔ܌ౢғǴڰۓ࣬ǵ

౽୏࣬Ϸྋ፦ޑឦ܄ٰዴۓǶќ΋Бय़Ǵڰۓ࣬ᆶࢬ୏࣬ޑམଛࢂߚதӭϡޑǴԶ Ъϩ݋ނҁي཮Ӣࣁ pH ॶׯᡂځ߄य़੝܄Ǵӵႝ಻ஏࡋϷ஥ႝ୷იޑ࣬ჹՏ࿼Ƕ ڰۓ࣬ޑว৖ϩࣁ׷਑ҁيᚆηූ୷ޑஏࡋǵҥᡏۓӛϷ੝܄Ǵх֖ϯᏢ่ᄬǵᛙ ۓ܄ǵϾሜ౗ǵᗭಈЁκჹܭ่ӝΚڀԖᒧ᏷܄Ǵ຾Զቹៜቫ݋ှ݋ࡋǶന߃Ҕа բࣁڰۓ࣬ޑࣁᠼᆢનǴೱௗΒΌ୷਽୷Ό✊ (diethylaminoethyl, DEAE) Ϸ♐Ҙ

୷ (carboxymethyl, CM)(Sober and Peterson, 1954; Sober et al., 1956)Ǵ౜Ϟаύ܄ᆫ ӝނӵጋᆒ (dextran)ǵᛏિ (agarose) Ϸӝԋᆫӝނӵᐋિ (resin) ࣁЬǶ

! ! ࣁၲډؼӳޑϩᚆϷගଯቫ݋ှ݋ࡋǴ፾྽ޑڰۓ࣬Ϸࢬ୏࣬ᒧ᏷ߚதख़ाǴ ϩ݋ނ่ӝܭڰۓ࣬ޑமࡋёҗᚆηமࡋǵࢬ୏࣬ pH ॶǵᚆηҬඤޑႝ಻மࡋϷ ϩ݋ނҁي܌௓ڋǶࢬ୏࣬ (܈጗ፂన) ޑᜪࠠǴх֖ణᚆηᐚࡋǴ཮ቹៜϩ݋ނ ޑࢲ܄Ϸྋှࡋ(Peterson and Torres, 1984; Cramer and Brooks, 1993)Ƕ

! ! ᚆηҬඤس಍ёϩࣁᗖ่Ϸѐᗖ่ޑၸำǴϩ݋ނᗖ่Կڰۓ࣬Ǵᙖҗࢬ୏࣬

аೱុ܈ఊࡋޑ pH ॶ܈ᡶᐚࡋ٬ϩ݋ނᆶڰۓ࣬ޑҬϕբҔ෧১Ǵ܈ޣуΕᆶڰ ۓ࣬Ԗ ଯᒃ کΚ ޑϩ ηӵ carboxymethydextran ǵDEAE-dextran ǵdextransulfate (Cramer and Brooks, 1993)ǵampholyte(Leaback and Robinson, 1975) Ǵၲډؑගϩᚆ ϐਏ݀Ƕ

4.1.1.ڰ

! ! ᚆηҬඤϐڰۓ࣬ڀԖٿঁ่ᄬǴ஥ႝ୷იୖᆶҬඤၸำǴаϷஒϐڰۓޑ୷

፦Ǵࣣ཮ቹៜቫ݋่݀Ƕ྽஥ႝ୷იޑႝ಻Ϸமࡋ،ۓϩ݋ނᗖ่ޑ੝౦܄ϷமࡋǴ Զ୷፦߾ቹៜނϯӼۓ܄ǵڰۓ࣬ޑࢬ୏੝܄Ϸߚ஑΋܄ᗖ่Ƕёၲډޑႝ಻மࡋ ϷᚆηҬඤޑ่ӝՏᗺڗ،ܭ୷፦ޑϯᏢ܄፦ᆶΟᆢ่ᄬǴԶᗭಈޑЁκελǵޔ ৩ϩѲϷϾሜࡋࣁ،ۓቫ݋ှ݋ࡋϐख़ाୖኧǶڰۓ࣬ёಉϩࣁѤᜪǴ১഍ᚆηǵ ம഍ᚆηǵ১໚ᚆηϷம໚ᚆηǶᚆηޑம১ᆶϩ݋ނᗖ่ޑமࡋคᜢǴ߄Ңځှ

ᚆำࡋǴமᚆηၨ১ᚆηૈӧၨቨޑ pH ॶጄൎύှᚆǴ΋૓ٰᇥǴ১഍໚ᚆη፾

ӝϐ pH ॶࣁ 5.5-9.5ǴԶம഍໚ᚆη߾ё፾ᔈ pH ॶ 2-12Ƕ

4.1.2.ࢬ୏࣬

! ! ᚆηҬඤس಍ϐ΋ख़ाୖኧࣁࢬ୏࣬ޑ pH ॶǴځణᚆηᐚࡋϷಔԋ཮ׯᡂϩ

݋ނᆶڰۓ࣬ޑᗖ่ૈΚǴ຾Զቹៜቫ݋ϐှ݋ࡋϷϩ݋ނޑ่ᄬᆶфૈֹ᏾܄Ƕ ӭኧس಍ёऐڙ༾ለϷ༾ᡵϐᕉნǴpH ॶ 6.0-8.5Ƕ౛གྷ௃ݩϐΠǴࢬ୏࣬٠ό཮

ᆶᚆηҬඤس಍຾ՉբҔǶ

4.2 ϩηᑔᒧቫ݋ݤ (size exclusion chromatography)

! ! ϩηᑔᒧቫ݋ݤǴёஒϩ݋ނ٩ϩηໆϩᚆǴ܈ѐନλϩηނ፦ǴӵᡶᜪǴၲ

ډપϯҞޑǶჹܭϩηᑔᒧቫ݋Ǵ٠ค಍΋ڮӜǴᏉጤၸᘠቫ݋ݤ (gel filtration chromatography, GFC)ǵᏉጤᅖ೸ቫ݋ݤ (gel permeation chromatography, GPC) Ϸ ϩηᑔᒧቫ݋ݤ (size exclusion chromatography, SEC) ࣣаඔॊ࣬ӕמೌǶϩᚆ่

״ࣁനλޑϩη೏ؑගрٰǴԶനεޑϩηၨϿ຾ΕϾࢰᡏᑈǴനӃ೏ؑගрٰǶ ϩηໆനεޑϩηคݤ຾ΕϾࢰύǴࡺ཮നӃ೯ၸᆅࢊǴ࿶җᆅࢊޜሜᡏᑈύࢬࢱ рٰǴջࣁᆅࢊޑ void volume (Vo)ǴԶനλޑϩη߾࿶җᆅࢊύ܌Ԗࢬࢱనᡏᑈ Զ೏ؑගрٰ (total accessible volume, Vt)Ǵ܌Ԗᡏᑈ (Vt) ջࣁᆅࢊᗭಈѦޑᡏᑈ

(Vo) ᆶᆅࢊᗭಈϣޑᡏᑈ (Vi)ǴVt= Vo + ViǶϩ݋ނޑᅉ੮ਔ໔җؑගᡏᑈۓໆǴ

V

̇

V

̇

΋ঁֹӄ௨ନϩηϐϩଛ߯ኧࣁ႟ǴԶ྽΋ϩη೯ၸ܌ԖϾࢰਔǴ

̇

V

̇

ᆅࢊЁκǴࡺϩଛ߯ኧ (

̇

კΜǵ܌Ԗᡏᑈ (Vt)ǵᆅࢊᗭಈѦޑᡏᑈ (Vo)ǵᆅࢊᗭಈϣޑᡏᑈ (Vi) ᆶᅉ੮ ᡏᑈ(Ve)ϐᜢ߯ҢཀკǶ

Figure 10. Sketch illustrating some of the most important terms used in size exclusion chromatography(Gooding and Regnier, 1990).

4.2.1. ڰ

! ! ΋૓٬Ҕϐጤᡏځ่ᄬࣁҬᖄ (crosslinked) ӭᗐ܈ӭ两Ǵځեᐒఓᛙۓ܄Ǵ คݤҔܭଯਏૈϩηᑔᒧቫ݋ (HPSEC)Ǵଯਏૈϩηᑔᒧቫ݋ሡा semi-rigid organic gel Ϸ rigid silica-based stationary phase མଛޔ৩λޑጤᡏᗭಈǴᓬᗺࣁ෧Ͽ ϩ݋ਔ໔ǴځϾ৩ЁκᆶᡏᑈǴό཮ڙډࢬࢱనׯᡂቹៜǴܭނ౛ᛙۓ܄Ϸϩ݋ਏ

ૈ΢ၨࣁᓬຫǶଯਏૈϩηᑔᒧቫ݋ጤᡏϾࢰЁκϟܭ 5-400 nm ၨத٬ҔǴࣁၲ

ډؼӳޑϩ݋Ǵᒧ᏷Ͼ৩ЁκϷځϩѲཱུࣁख़ाǶᆶᆫӝ׷਑ϐጤᡏКၨǴа silica ࣁЬޑ׷਑ჹᓸΚϷ pH ॶၨࣁ௵གǶࣁլܺ১ᚆηҬϕբҔϷᆢ࡭ᛙۓ܄Ǵsilica а diol (1,2-dihydroxy-3-propoxyprolyl bonding) অႬǴঅႬࡕጤᡏჹߎឦ਼ϯނᛙ ۓǴ٠ёऐڙ pH ॶډ 8.5ǶϾࢰᡏᑈК౗ (Vi/Vo) ޔௗቹៜᆅࢊϐှ݋ࡋǶ

4.2.2.౽

! ! ౛གྷޑࢬ୏࣬ό཮ቹៜϩηᑔᒧس಍Ǵ٣ჴ΢ǴྋᏊϣ৒ࠅ཮ቹៜϩᚆ่݀Ǵ ቹៜϩ݋ނϷڰۓ࣬໔ޑҬϕբҔǴςவ silica-based ጤᡏளډ᛾ჴǶᗖ่ܭ silica Ѩ௳தӢ steric ϐ౛җǴࡺ׷਑೯தϝԖ஥ॄႝޑ silanol ୷იǴ٠คঅႬԋ১҅ႝ

ޑҬඤᕉნǴԶԜᚆηҬϕբҔϐቹៜёҗׯᡂ pH ܈уΕᡶᜪፓ᏾Ƕ

4.2.2.1. ׯᡂ pH ॶ

! ! Silanol ූ୷ pK ॶࣁ 3.5 Կ 4Ǵࣁ΋১ለ୷იǴ྽నᡏᆶϩ݋ނޑ pH ॶόӕǴ

߾཮ቹៜᚆη໔ҬϕբҔǴ΋ѿࢬ୏࣬ϐ pH ॶׯᡂǴϩ݋ނϐ pH ॶΨёૈׯᡂǶ

஥҅ႝޑϩ݋ނܭ፾྽ޑ጗ፂన (pI>pH) ύ཮ᅉ੮ܭᚆηҬඤس಍ύǴၨႣයа

ၨଯޑ Ve (ᅉ੮ᡏᑈ) ೏ؑගрٰǶϩ݋ނऩ஥ॄႝǴᆶ஥ॄႝޑᚆηҬඤس಍ϕ ѾǴ཮זೲ೏ؑගрٰǶ

4.2.2.2. ׯᡂᡶᐚࡋ

! ! ᚆη໔ҬϕբҔёᙖҗуΕύ܄ᡶᜪፓ᏾Ƕࢬ୏֖࣬Ԗଯܭ 0.6 M ޑᚆηம ࡋǴ཮ቚу౧НբҔǴ೷ԋϩ݋ނᅉ੮Ǵऩᐚࡋλܭ 0.1 M ߾཮ᔅշϩ݋ނϩᚆǴ

ࡺ΋૓٬Ҕϐᡶᐚࡋϟܭ 0.1 Կ 0.5 MǴԶΒሽᚆηޑᚆηமࡋࣣଯܭ΋ሽᚆη (Rogner, 1999)Ƕ

ಃ

ಃѤകǵġխࣝᔠෳБݤ

ಃ΋࿯ġǵ୷ᘵϟಏ

! ! ໺಍ϯᏢϩ݋Бݤࣁவወӧυᘋނ፦ύϩᚆǵൂᚆǴϷۓໆǵ᠘ۓБݤ (Biagini et al., 2004)Ƕа۳ޑБݤڀԖ೚ӭલᗺǴхࡴଯࡋമ୏Ϸܳ຦೛ഢЍр (ӵ

਻࣬ቫ݋ (GC)ǵన࣬ቫ݋ (LC)ǵ፦᛼ሺ (MS) ܈Սᖄϩ݋ (GC-MSǵLC-tandem- MS)ǶӆޣǴ໺಍ϩ݋Бݤܭϩᚆપϯޑӣԏ౗٠όᛙۓǴ٠ӧ೚ӭ௃ݩΠౢғس

಍ᇤৡ(Baker et al., 2000; Barr et al., 2002; Carabias-Martınez et al., 2003)Ƕ໺಍ϯᏢ ϩ݋ޑඹжБݤࣁխࣝϩ݋ݤǴ੝ձࢂሇનխࣝϩ݋ (enzyme immunoassays, EIAs) Ϸሇનೱ่խࣝ֎ߕ၂ᡍ (Enzyme-linked immunosorbent assay, ELISA)Ǵதـܭᖏ

׉ບᘐෳໆǵᛰނᑔᒧǵᕉნҔᛰϩ݋(Biagini et al., 1993; Biagini et al., 1995; Biagini

et al., 2004)Ƕ

ಃΒ࿯ġǵלচᆶъלচᅿᜪ

! ! २ԛ ELISA р౜ܭ 1976 ԃǴҞ߻ჹܭғނ৮܀ᇙᏊٯӵࣅੴ዗ (anthrax) ޑ

ෳໆς೏᛾ჴԖਏǶխࣝϩ݋ࣁෳۓלচϷלᡏፄӝނޑౢғǶלচ΋૓ࣁεϩηǴ ӵೈқ፦ǵӭᗐǵਡለǴڈᐟౢғխࣝϸᔈǴځдނ፦ӵᛰࠔǵၭᛰ฻ǴӢϩηໆ ϼλคݤൂᐱբࣁלচǴѸ໪ᆶεϩηၩᡏೱ่ (೯தࣁೈқ፦) аౢғלচ܄٠ ЇวխࣝϸᔈǴԜλϩη߾ᆀࣁъלচ (hapten)Ǵ೚ӭᕉნᇙᏊ (ӵၭᛰ܈ၭᛰж ᖴނ) ࣁъלচǶҔܭբࣁъלচ-ೈқ፦Ӆ೫לচޑೈқ፦ၩᡏǴځᒧ᏷ߚதख़

ा(Engvall and Perlmann, 1972; Striley et al., 1999)Ǵೱ่ܭၩᡏޑъלচኧҞϷӅ ೫ނޑϸᔈࣣ཮ቹៜלᡏޑᒃکΚǴъלচޑપࡋҭ࣬ӕख़ाǴёૈ཮Ꮴठߚ੝౦

܄לᡏ׎ԋǶ໔႖ϩηதҔܭъלচޑᇙഢǴаቚуӅ೫ϐъלচޑלᡏ஑΋܄Ƕ לᡏᆶלচ܈ъלচᗖ่ޑૈΚҗଛᡏ (ligand) ᆶלᡏܭᗖ่Տ࿼ޑ่ᄬکϯᏢ բҔ܌௓ڋǴלচϷלᡏ໔բҔࣁё଍ǴЪคӅሽᗖୖᆶ(Dankwardt, 2000)ǶԜբ Ҕҗ፦ໆբҔۓࡓ௓ڋǶ

Ag+Ab=AgAb Ϸ

 ൌ ‰„

ሾ‰ሿሾ„ሿ‘Ž^ିଵ

K ࣁᒃکதኧǴAgAb ߾ࣁלচǵלᡏፄӝނǶଯᒃکதኧࣁமלচǵלᡏբҔǴ Ъܭխࣝϩ݋ύԖၨեᔠෳཱུज़ (limits of detection, LOD)Ƕ

ಃ

ಃΟ࿯ġǵלᡏᅿᜪ

! ! থ٢୏ނޑխࣝس಍ౢғϖᅿόӕᜪձޑלᡏǴIgAǵIgDǵIgEǵIgGǵIgMǶ խࣝౚೈқڀԖٿచ࣬ӕޑख़᜘ (heavy chain, 50-60 kDa) ᆶᇸ᜘ (light chain, ~25 kDa)Ƕख़᜘ᆶᇸ᜘ࣣԖᒿלᡏόӕԶׯᡂ่ᄬޑᡂ౦୔ (VH Ϸ VL)Ǵջࣁᆶלচ ᗖ่ೀǶٿచ᜘ځᎩ೽ϩ೏ᆀࣁࡡۓ୔ (CH Ϸ CL)Ǵځữ୷ለ่ᄬᗲϿׯᡂǴԜ ׯᡂ،ۓΑᇸ᜘ޑ٥ࠠ (ĸǵλ) Ϸख़᜘ޑᅿᜪ (α, δ, γ, ε, μ)Ƕࢌ٤לᡏ߾ᙖҗࡡۓ

୔ᆶಒझᗖ่ǶIgG ࣁӭኧথ٢୏ނޑᓬ༈לᡏᅿᜪǴࢂࣁҔаว৖ EIA ޑЬा

לᡏǴԶ ELISA ё٬Ҕൂਲ਼܈ӭਲ਼לᡏǶӭਲ਼לᡏࣁஒלচᆶ՘ᏊݙΕ୏ނ (೯ தࣁټη)Ǵԏ໣ځՈమԶள(Hines et al., 2003)Ǵஒϐ຾΋؁પϯϩᚆǴౢғ஑΋܄

ӭਲ਼לᡏ(Khan et al., 2003)Ƕӭਲ਼לᡏӵځӜǴࣁխࣝౚೈқϐషӝނǴଞჹלচ

΢ӭᅿלচ،ۓՏ࿼ (epitope)Ƕဍዦಒझਲ਼Ϸӝԋלᡏޑ๠᠌ B ಒझᑼӝԋᑼӝ ዦಒझǴౢғϐלᡏ໻ڀ΋לচ،ۓՏ࿼Ǵࡺᆀൂਲ਼לᡏǶൂਲ਼לᡏගٮ΋ೱុЪ ڀ஑΋܄ޑᔠෳኳԄ(Trout et al., 2004)Ƕܫ৔խࣝϩ݋ݤ (Radioimmunoassay, RIA)

٬Ҕܫ৔܄኱૶ (ӵ iodine 125Ǵ¹²⁵I)ǴෳۓלচϷלᡏ໔ϸᔈǶלচǵלᡏϸᔈ߄

౜߾٬Ҕ՚ᅦीኧෳۓ(Biagini et al., 1985)Ƕӭኧ RIA ς೏ ELISA ڗжǴԖਔΨᙁ ᆀࣁ EIAǶ

ಃ

ಃѤ࿯ġǵሇનೱ่խࣝ֎ߕ၂ᡍ (Enzyme linked immunosorbent assay)!

4.1 ڰۓ࣬ (solid phase)

! ! ELISA ࢂஒלচ܈לᡏа጗ፂనีញࡕ༡ܭ΋ڰۓ࣬Ǵচ౛ࢂа೏୏֎ߕ (passive adsorption) ஒϸᔈނڰۓܭ΋߄य़ǴԜᗖ่௢ෳࣁ౧НբҔ

(hydrophobic interaction)ǵᓉႝЇΚ (electrostatic attraction) ϷΥቺґᅟΚ (van der Waals forces)ǶҞ߻ၨத٬Ҕ 96-well polystyrene microtiter plateǴሽ਱եǵܰ٬ҔǴ ٠೴ᅌว৖Ծ୏ϯ(Butler et al., 1992) Ƕ

4.2 ߔ༞ (blocking)

! ! ჴᡍၸำύǴԖΟᅿ௃ݩ཮Ꮴठߚ஑΋܄ᗖ่Ǵϩձࢂ (1) לচǵלᡏϷ୷፦

໔ޑߚ஑΋܄ϸᔈǴ(2) ߚ஑΋܄ᚇ፦ (Ҭΰ) ԡࢉלচϷלᡏϷ (3) όܴԡࢉ

(Tsang et al., 1985b)Ƕࣁ෧Ͽߚ஑΋܄ᗖ่Ǵ٬Ҕ tris (hydroxymethyl) aminomethaneǵ ethanolamine ࣣஒೱௗՏ࿼ (binding site) ߔ༞ (blocking)ǵѐࢲϯ (inactivate)Ǵа फ़եङඳॶ(Renert et al., 1979)ǴΨёа٬ҔځдྋనӵФՈమқೈқ (bovine serum albumin , BSA)(Towbin et al., 1979; Aubertin et al., 1983)ǵfetal bovine serum(De Blas and Cherwinski, 1983; Ramirez et al., 1983)ǵhemoglobin (Gershoni and Palade, 1982)ǵgelatin(Lim and Kasamatsu, 1983)ǵTween 20(Battaiger et al., 1982; Muilerman

et al., 1982; Wedege and Svenneby, 1986)ϷФѪ(Johnson et al., 1984)Ƕ

4.3 ևՅᏊ

! ! ᔠෳس಍೯தࣁሇનೱ่ԿϸᔈނǴதҔޑևՅᏊԖ PNPP (p-nitrophenyl phosphate)Ǵᔠෳ alkaline phosphataseǴౢғ໳ՅНྋ܄ౢނǴෳۓ 405 nm

֎ӀॶǶABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid))ǵOPD (o-phenylenediamine dihydrochloride) ᆶ HRP (horseradish peroxidase) ϸᔈϩձౢғ ᆘՅǵ໳ՅНྋ܄ౢނǴ୤Ԗ TMB (3,3',5,5'-tetramethylbenzidine) ᆶ HRP ϸᔈౢ

ғϐᙔՅނ፦཮ӧуΕ౷ለ܈ᕗለಖЗϸᔈࡕᙯࣁ໳ՅౢނǶᔠෳϐᡫ௵ࡋа TMB ന٫Ǵၨځдڙ፦৒਼ܰϯǴࡺёזೲևՅǶ

4.4 ELISA Б

! ! ELISA ёаӭᅿБԄ߄౜Ǵޔௗǵ໔ௗǵਂਆϷᝡݾ฻Ƕޔௗ ELISA

(კΜ΋) ࣁ୷ҁ ELISA БԄǴஒ΋ϩ݋ނᗖ่Կ༾Ͼዬ߄य़ǴуΕჹϩ݋ނڀ஑

΋܄ᆶൔᏤس಍ޑלᡏϷևՅᏊ٬ϐϸᔈǴғԋևՅނ፦Ƕ໔ௗ ELISA ϩ݋ݤǴ ϩ݋ނӵъלচϷלচӕኬ֎ߕԿ༾Ͼዬ߄य़Ǵ٩ׇуΕჹϩ݋ނڀ஑΋܄ᗖ่

ϐ߃ભלᡏϷჹ߃ભלᡏڀ஑΋܄ϐΒભלᡏǴᆶևՅᏊϸᔈࡕаϩӀӀࡋीෳ

ۓ֎ӀॶǴևՅໆ҅КܭᆶΒભלᡏೱ่ໆǶ

კΜ΋ǵޔௗϷ໔ௗխࣝ֎ߕ၂ᡍǶ

Figure 11. Direct and indirect immunoassay. In a direct assay (a), analyte (hapten, Ab, Ag) is bound to a solid support (i.e., bead or microplate). Reporter labeled Ab is introduced to the immobilized analyte, forming an Ag-Ab complex. After washing, the concentration of analyte is measured by radiometric, colorimetric, or fluorometric detection of the reporter system. In an indirect format (b, c), a primary Ab specific for

the analyte is introduced to the solid support bound analyte. After washing, a secondary labeled Ab, specific for the primary Ab, is added to the system. The concentration of analyte is measured by radiometric, colorimetric, or fluorometric detection of the reporter system(Biagini et al., 2006).

! ! ELISA ΨԖਂਆኳԄ (capture formate) (კΜΒ)Ǵלচਂਆ ELISA ݤ (ΞᆀΟ

ܴݯݤ)Ǵࣁלচ೏੝౦܄לᡏਂਆǴ֎ߕܭ༾Ͼዬ߄य़ǴуΕڀ஑΋܄ޑ኱૶ל ᡏϷևՅᏊǴ٬ҔϩӀӀࡋीෳۓځ֎ӀॶǶନԜϐѦǴᝡݾ (competitive) ELISA

߾ࢂϩ݋ނᆶ኱૶ϩ݋ނ໔ᝡݾᗖ่Ƕ྽ϩ݋ނᐚࡋၨଯǴ኱૶ϩ݋ނᐚࡋၨեਔǴ ߞဦ෧১ǴԜϩ݋Бݤ࿶অႬࡕǴࣁ blocking ELISAǴஒ҂኱૶ޑϩ݋ނӃܭ኱૶

ϩ݋ނуΕǶ

კΜΒǵਂਆϷᝡݾխࣝ֎ߕ၂ᡍǶ

Figure 12. Capture and competitive immunoassay. In an Ag capture (sometimes called sandwich) assay,Ab, specific for the analyte, is bound to a solid support. Added analyte is bound by the first specific Ab (a).After washing, another labeled Ab, specific for another epitope on the Ag, is added (b). Concentration of analyte is measured by radiometric, colorimetric, or fluorometric detection of the reporter system. In a competitive assay, analyte and a reporter labeled analyte are allowed to compete for binding sites with the immobilized antibodies (c) and bind in relation to their relative

colorimetric, or fluorometric detection of the reporter system. With

higher concentrations of analyte, less of the labeled analyte is bound yielding reduced signal(Biagini et al., 2006).

ಃ

ಃϖ࿯ġǵൂਲ਼לᡏ

! ! Georges Köhler ک Cesar Milstein ܭ 1975 ԃว৖рᇙഢൂਲ਼לᡏמೌǶаל চխࣝλႵǴϩᚆр๠ಒझǴஒғౢלᡏޑ B ಒझᆶମᡎዦಒझᑼӝǴౢғᑼӝ ዦಒझǴ຾Չज़ኧีញ୻ᎦǴ׎ԋൂ΋ಒझਲ਼Ǵ໻ғԋᒣᇡ୤΋לচ،ۓՏ࿼

(epitope) ޑלᡏǴջࣁൂਲ਼לᡏ(Köhler and Milstein, 1975)Ƕ

5.1 LM2 ൂਲ਼לᡏ (anti-AGII monoclonal antibody)

! ! LM2 ࣁᒣᇡ AGII ݀ጤӭᗐ ß-linked glucuronic acid ่ᄬޑൂਲ਼לᡏǶᇙഢБ Ԅࣁஒዿԯ (Oryza sativa L. cv. Moroberekan) ಒझਲ਼୻ᎦనࣁלচǴ؂ԛа 100 μg མଛ٢Ꮚ (Freund’s adjuvants) խࣝεႵ (Wistar Rat)ǶࡑՈమਏሽଯਔǴڗځ๠᠌

ϐ B ಒझᆶମᡎዦಒझ IR983F ຾Չಒझᑼӝࡕज़ኧีញ୻ᎦǶ٬Ҕሇનೱ่խ

ࣝ֎ߕݤ (enzyme-linked immunosorbent assayǴELISA) բࣁ߃ԛᑔᒧǴᗺᅄݤ (dot blots) ǵ Ջ Б Ꮐ ᗺ ݤ (Western blot) ᆶ ਥ Ӿ ಒ झ ϐ խ ࣝ ᑻ Ӏ ࢉ Յ Ǵ ᑔ ᒧ р LM2(Smallwood et al., 1996) Ƕ ל চ ، ۓ Տ ࿼ ࣁ à-linked glucuronic acid Ǵ ࢂ arabinogalactan type II (AGII) ΢ arabinogalactan ޑڗж୷Ǵࡺ LM2 ೏ᇡࣁᒣᇡ arabinogalactan type II (AGII) ޑלᡏǶ

5.2 LM5 ൂਲ਼לᡏ (anti-(1ʈ4)-ß-D-galactan monoclonal antibody)

! ! LM5 ࣁᒣᇡ AGI ݀ጤӭᗐ (1ʈ4)-ß-D-galactan ่ᄬޑൂਲ਼לᡏǶᇙഢБԄࣁ પϯพन (Lycopersicon esculentum cvs Ailsa Craig and Solairo) ϐಒझᏛӭᗐǴᆶ Ҙ୷ϯФՈమқೈқ (bovine serum albuminǴBSA) ᇙԋᑗೈқ (neoglycoprotein)Ǵ բࣁלচխࣝεႵ (Wistar Rat)ǴࡑՈమਏሽଯਔǴڗځ๠᠌ϐ B ಒझᆶମᡎዦಒ

झ IR983F ຾Չಒझᑼӝࡕज़ኧีញ୻ᎦǶ٬Ҕሇનೱ่խࣝ֎ߕݤ (enzyme- linked immunosorbent assayǴELISA) Ϸխࣝᗺᅄݤ (immunodot-binding assay)Ǵᑔ ᒧр LM5 (Jones et al., 1997)Ƕ݀ጤύڀԖၨӭԜ่ᄬࣁ arabinogalactan type IǴ܌

а LM5 ೏ᇡࣁᒣᇡ arabinogalactan type I (AGI) ϐלᡏǶ

5.3 LM6 ൂ

! ! LM6 ࣁᒣᇡ RGI ݀ጤӭᗐϩЍ linear-(1ʈ5)-α-L-arabinan ่ᄬޑൂਲ਼לᡏǶ ஒ (1→5)-α-L-arabinoheptose ᆶ BSA ᇙԋ neoglycoprotein---Ara7-BSAǴ؂΋ವᅟ (mol) ೈқ፦΢ڀԖ 3 ঁ hepatasaccharides ჲᑗǶ؂ԛа 200 μg མଛ٢Ꮚ (Freund’s adjuvants) խࣝεႵ (Wistar Rat)ǴࡑՈమਏሽଯਔǴڗځ๠᠌ϐ B ಒझᆶମᡎዦ ಒझ IR983F ຾Չಒझᑼӝࡕज़ኧีញ୻ᎦǶ٬Ҕሇનೱ่խࣝ֎ߕݤ (enzyme- linked immunosorbent assayǴELISA) Ϸխࣝᗺᅄݤ (immunodot-binding assay)Ǵᑔ ᒧр LM6 (Willats et al., 1998)Ƕ

5.4 LM7 ൂਲ਼לᡏ (anti-homogalacturonan monoclonal antibody)

! ! LM7 ࣁᒣᇡ݀ጤӭᗐ homogalacturonan ่ᄬޑൂਲ਼לᡏǶᇙഢБԄࣁа lime pectin ଺ࣁלচǴځҘ୷✊ϯำࡋ (degree of methyl-esterification, DE) ࣁ 22.9%Ǵ ㄽữϯำࡋ (degree of amidation) ࣁ 27.3%Ǵѳ֡ϩηໆࣁ 84 kDaǶམଛ٢Ꮚ (Freund’s adjuvants) խࣝεႵ (Wistar Rat)ǴࡑՈమਏሽଯਔǴڗځ๠᠌ϐ B ಒझ ᆶମᡎዦಒझ IR983F ຾Չಒझᑼӝࡕज़ኧีញ୻ᎦǶ٬Ҕሇનೱ่խࣝ֎ߕݤ (enzyme-linked immunosorbent assayǴELISA) Ϸխࣝᗺᅄݤ (immunodot-binding assay)Ǵᑔᒧр LM20(Verhertbruggen et al., 2009; Marcus et al., 2010)Ƕ

5.5 LM10 ൂਲ਼לᡏ (anti-(1ʈ4)-ß-D-xylan monoclonal antibody)

! ! LM10 ࣁ ᒣ ᇡ ݀ ጤ ӭ ᗐ AGII ϐ (1 ʈ 4)-ß-D-xylan ่ ᄬ ޑ ൂ ਲ਼ ל ᡏ Ƕ ஒ

xylopentaose (X5) ᆶ BSA ᇙԋᑗೈқ (neoglycoprotein)Ǵ٬؂΋ঁ BSA ϩη΢ڀ Ԗ 14 ঁ xylopentaose ჲᑗǶ؂ԛа 100 μg མଛ٢Ꮚ (Freund’s adjuvants) խࣝε Ⴕ (Wistar Rat)ǴࡑՈమਏሽଯਔǴڗځ๠᠌ϐ B ಒझᆶମᡎዦಒझ IR983F ຾Չ ಒ झ ᑼ ӝ ࡕ ज़ ኧ ี ញ ୻ Ꭶ Ƕ ٬ Ҕ ሇ ન ೱ ่ խ ࣝ ֎ ߕ ݤ (enzyme-linked immunosorbent assayǴELISA) Ϸխࣝᗺᅄݤ (immunodot-binding assay)Ǵᑔᒧр LM10 (McCartney et al., 2005)Ƕ

5.6 LM19 ൂ

! ! LM19 ࣁᒣᇡ݀ጤӭᗐ homogalacturonan ่ᄬޑൂਲ਼לᡏǶᇙഢБԄࣁа apple fruitǴམଛ٢Ꮚ (Freund’s adjuvants) խࣝεႵ (Wistar Rat)ǴࡑՈమਏሽଯਔǴڗ ځ๠᠌ϐ B ಒझᆶମᡎዦಒझ IR983F ຾Չಒझᑼӝࡕज़ኧีញ୻ᎦǶ٬Ҕሇનೱ

่ խ ࣝ ֎ ߕ ݤ (enzyme-linked immunosorbent assay Ǵ ELISA) Ϸ խ ࣝ ᗺ ᅄ ݤ (immunodot-binding assay)Ǵᑔᒧр LM19(Verhertbruggen et al., 2009; Marcus et al., 2010)Ƕ

5.7 LM20 ൂਲ਼לᡏ (anti-homogalacturonan monoclonal antibody)

! ! LM20 ࣁᒣᇡ݀ጤӭᗐ homogalacturonan ่ᄬޑൂਲ਼לᡏǶᇙഢБԄࣁа

Arabidopsis thaliana ޑ

5.8 JIM7 ൂਲ਼לᡏ (anti-homogalacturonan monoclonal antibody)

! ! JIM7 ࣁᒣᇡ݀ጤӭᗐ homogalacturonan ่ᄬޑൂਲ਼לᡏǶᇙഢБԄࣁа Carrot

(Daucus carota L. cv. Early Nantes) ᅿη଺ࣁלচǴམଛ٢Ꮚ (Freund’s adjuvants) խࣝεႵ (Wistar Rat)ǴࡑՈమਏሽଯਔǴڗځ๠᠌ϐ B ಒझᆶମᡎዦಒझ IR983F

຾ Չ ಒ झ ᑼ ӝ ࡕ ज़ ኧ ี ញ ୻ Ꭶ Ƕ ٬ Ҕ ሇ ન ೱ ่ խ ࣝ ֎ ߕ ݤ (enzyme-linked immunosorbent assayǴELISA) Ϸխࣝᗺᅄݤ (immunodot-binding assay)Ǵᑔᒧр JIM7(Knox et al., 1990; Willats et al., 2000; Clausen et al., 2003)Ƕ

5.9 ൂ

! ! ᒣᇡ੝ۓಒझ߄य़ᗐᜪלচ،ۓՏ࿼ (epitope) ޑൂਲ਼לᡏ (MAbs) ࣁӭф

ૈϩη௖ଞǴҔܭ෌ނಒझǵϩηғނϷಔᙃว৖Ƕ1970 ԃ२ԛа޸ણᏛϷᝯӭ ᗐࣁჹຝࣴزځխࣝۓՏ(immunolocalization)(Knox et al., 1970; Vreeland, 1970)Ǵ٠ Ъаൂਲ਼לᡏ຾Չ෌ނխࣝಒझϯᏢϷ޸ણޑלচϷၸ௵চࣴز (Knox et al., 1980)Ǵӧ 1980 ԃ໒ۈᜢܭ੝ۓಒझϐӭᗐלՈమࣴز(Moore et al., 1986; Cassab and Varner, 1987; Stafstrom and Staehelin, 1988; Ryser and Keller, 1992; Condit, 1993;

Swords and Staehelin, 1993)Ǵൂਲ਼לᡏࣁ΋ԖΚπڀǴගٮ෌ނಒझ่ᄬǵಔᙃϷ фૈၗૻǴӵಒझᏛᆶझѦ୷፦ޑಔᙃϷ่ᄬǵКၨόӕಒझ໔วػৡ౦ǵዴۓಒ झᏛᜢܭᒪ໺ӭኬ܄ޑϩηৡ౦ǵ௖ෳಒझᏛ܈ᆫӝނޑלচ،ۓՏ࿼ϐфૈǴ٠ ڀԖᐱ੝ޑᓬ༈ӵ஑΋੝ۓ܄ (monospecificity)Ǵᒣᇡ҂પϯϐ෌ނ๧ڗނ੝ձԖ Ҕ(Anderson et al., 1984)Ƕ஑΋੝ۓ܄ჹܭᗐೈқϷӭᗐཱུࣁख़ाǴӢࣁ೭ᜪלচ ೯தୖᚇځдלচ،ۓՏ࿼Ǵࡺሡा஑΋੝ۓޑӭਲ਼לՈమપϯϐǶ෌ނಒझ߄य़ ڀԖӭኬϯಒझ୔ୱ (domains)Ǵх֖ಒझጢϷಒझᏛǴගٮಒझว৖фૈ(Roberts, 1989; 1990)ǴҞ߻ൂਲ਼לᡏҔܭ೚ӭϩ݋Бय़ӵᗖ่ǵғϯ੝܄ᆶ෌ނಒझ߄य़ޑ ว৖ፓ௓ǵຑ՗ಒझጢપϯำࡋǵচғ፦ᡏ੝܄ (protoplast properties)ǵ᠘ۓಒझ ߄य़ಔԋϷܭಔᙃکಒझޑϩѲኳԄǴԖճܭᔠෳϩϯלচǶ

! ! аൂᗐ܈ჲᗐᝡݾൂਲ਼לᡏ-לচޑ ELISA ᔠෳҭёࡰр੝ۓޑלচ،ۓՏ

࿼(Pennell et al., 1989; Pennell et al., 1991)Ǵջ٬ъלচ٠คᡉҢҺՖԖᜢלচ่

ᄬǴҭёவխࣝೱ่ύளޕځᄬࠠϷ࣬ᜢಔԋ (კΜΟ) (Pennell et al., 1989)Ƕ

კΜΟǵൂਲ਼לᡏᒣᇡלচ،ۓՏ࿼ (epitope) ௢ෳӭᗐ่ᄬǶ

Figure 13. Monosaccharide components of carbohydrate epitopes. The binding

inhibition was determined by ELISA. The inhibition by gum arabic (a), an AGP, shows that MAC 207 and JIM8 recognize AGP epitopes. Further, the different patterns of inhibition by hapten monosaccharides (a) show that the MAC 207 and JIM8 epitopes are different, and that the MAC 207-reactive epitope probably contains arabinose and glucuronic acid. Chemical analysis of AGP carbohydrates suggests that the arabinose is a terminal, so it is likely that the MAC 207-reactive epitope is terminal as well (b). The composition of the JIM8 epitope is unknown but, as shown in (b), it is probably

subterminal. The AGP backbone is composed of 1,6-linked galactosyl residues. The numbers in the table refer to the inhibitor concentration required for 50% diminution in ELISA signal (Iso).

! ! Arabinogalactan-protein ࣁ΋ೈқӭᗐǴϩѲܭ෌ނಔᙃǴڀԖխࣝᡉ܄

(immunodominant)ǴࣁЬाౢғխࣝϸᔈޑ෌ނ่ᄬ(Anderson et al., 1984; Norman

et al., 1986; Evans et al., 1988; Knox et al., 1989; Pennell et al., 1989; Norman et al.,

ൂਲ਼לᡏς೏ᑔᒧىаܢל෌ނಒझጢ ATPase(Chin, 1982)ǵ׭ڋғߏન (auxin) ޑཱུ܄ၮᒡ (polar transport)(Jacobs and Gilbert, 1983) Ϸጋણቫচғ፦ᡏύ㱏ပለ

(abscisic acid) ϸ ᔈ Ƕ ӭ ᅿ ಒ झ Ꮫ ᆫ ӝ ނ ޑ ל Ո మ ܢ ל Ӛ ᅿ س ಍ ύ ᇨ Ꮴ ՜ ߏ (elongation) ޑ ғ ߏ ન Ǵ ӵ ҏ ԯ ख ޵ ᓋ (maize coleoptiles) ޑ ל ဟ ᆫ ᑗ 䁙 (antiglucanase)ǵᆘل΢खື (bean epicotyls) ޑלЕᆫᑗ䁙 (antixyloglucan)ǴϷᡳ

቏ل (chickpea) ޑל ß ъ٢ᑗ䁙 (anti-ß-galactosidase)(Hoson et al., 1991; Inouhe and Nevins, 1991; Hoson et al., 1992; Valero and Labrador, 1993)Ƕᒿ๱ଯᒃکΚ௖ଞ Ϸख़ಔ DNA ޑמೌୢШǴಒझᏛӭᗐϷᗐೈқޑלচ،ۓՏ࿼೴ᅌዴҥǶ

! ! ݀ጤࣁ΋ፄᚇӭᗐǴԖӭᅿ่ᄬ୔ୱ (domain)Ǵх֖ acidic homogalacturonanǵ rhamnogalacturonan Ϸύ܄ᗐЍ᜘(O'Neill et al., 1990)Ǵځфૈࣁύጤቫಒझᗹ๱ǵ

௓ڋ߃ભಒझᏛᚆηރᄊϷϾሜ౗ǵቹៜሇનϷځдᆫӝނբҔǴԶ݀ጤӭᗐТࢤ

߾ڀٛᑇᐒڋǶᙖҗ݀ጤϐխࣝۓՏว৖לᡏ௖ଞ (antibody probe)ǴЬाҔܭխ

ࣝᑻӀ (immunofluorescence) Ϸխࣝߎ (immunogold) ࣴزǶ݀ጤלᡏჹܭಒझϩ ϯԖӭᅿᔠෳБݤǴҔܭಒझǵ୻Ꭶ୷฻س಍(Van Engelen et al., 1991; Stephenson and Hawes, 1994; David et al., 1995; Stacey et al., 1995)ǴԜѦϝԖխࣝᒃکמೌ่

ӝॄࢉႝηᡉ༾᜔ᔠෳ(McCann et al., 1992)Ƕ᏾ᡏٰᇥǴၮҔ݀ጤלᡏёа߄౜ό ӕಒझᅿᜪǵಔᙃǵᏔ۔ǵᅿᜪύ݀ጤ✊ϯޑׯᡂǴᆶ݀ጤלᡏೱௗᆶցǴ٠όж ߄݀ጤр౜ԖคǴҗלচ،ۓՏ࿼ёаளޕಒझᏛ΢ࢂցڀԖঅႬ୷ǴҞ߻٬Ҕς ۓက (defined) ௖ଞෳۓҘ୷✊ϯТࢤǵrhamnogalacturonan ମࢎޑ੝ۓჲᑗׇӈ Ϸ arabinogalactan Ѝ᜘Ƕלᡏ௖ଞჹܭ݀ጤ่ᄬှ݋ཱུࣁख़ाǴёޕځԋߏวػޑ ቹៜୖኧǶғԋӭᗐלᡏޑख़ा٩ᏵࣁൂᗐϷჲᑗ ᆶೈқ፦ጠӝ׎ԋխࣝচ (immunogen)Ǵ٣ჴ΢ࢌ٤ӭᗐ٬Ҕ࣬ӕޑಔԋӵൂᗐϷᗖ่੝ቻǴԶ೭ᜪޑלՈ మ٠όڀ஑΋܄ǴᖐٯٰᇥǴ٬Ҕα-L-arabinofuranoside-arninophenyl protein բࣁ לচౢғޑלᡏёаᒣᇡ arabinoxylan Ϸ arabinogalactan(Kaku et al., 1986; Misaki

et al., 1988)ǴԜБݤς೏ҔܭౢғӵԛભಒझᏛύޑ xylanǵلᜪಒझ݈΢ޑ callose

ೈқ፦όڀሇનࢲ܄Ǵ໻ගٮಒझᏛ่ᄬբҔ(Showalter, 1993)Ƕ

! ! Arabinogalactan-protein ቶݱӸӧ෌ނύǴୖᆶಒझวػၸำǶAGP לᡏЬा

ᒣᇡҗ arabinogalactan ಔԋϐלচ،ۓՏ࿼Ǵ೏ຎࣁ෌ނύගٮխࣝϸᔈޑЬाՏ

࿼ǶКၨᝌੌ୻ᎦޑዿԯಒझϷचᡀጱಒझਲ਼Ǵϩᚆዿԯύ੝ۓϷߚ੝ۓ AGP ൂ ਲ਼לᡏǶLM2 ࢂҗዿԯౢғϐൂਲ਼לᡏǴӧ੝ۓ୻Ꭶ୷ύǴLM2 ᆶٿᅿ AGP ڀᒃ کϸᔈǴࠅѝᆶचᡀጱϐ΋ᅿ AGP ౢғᒃکϸᔈǶAGP ќ΋ख़ा੝ቻᆶಒझጢ࣬

ᜢǴಒझጢᆶНྋ܄झѦ AGP ϐ࣬ᜢ܄ё٬Ҕ LM2 ٰղۓǴᡉҢዿԯಒझጢ΢

AGP ࣁ౧Н܄ǴԶचᡀጱϐ AGP ߾ࣁᒃН܄(Smallwood et al., 1996)Ƕ

! ! ෌ނಒझᏛ่ᄬς࿶೴ᅌҗࣴزளޕǴಒझᏛޑϩη୔ୱ (molecular domain) Ϸಒझϩϯ೷ԋಒझᏛǵಒझጢ໔ϐৡ౦Ƕלᡏჹܭ෌ނ߄य़ޑ౛ှගٮཱུεޑଅ

᝘Ǵ߈ԃٰלᡏޑว৖׳җಒझᘉεډ୷ӢቫભǶ౜Ϟמೌёᙖҗ௓ڋ DNA ჹᔈ ϐጓዸלᡏ่ӝՏ࿼Ǵ೛ीᑔᒧϩη௖ଞǴቚу٬Ҕܭ෌ނಒझ߄य़ϐёૈ܄ǶҞ

߻ϝሡளޕಒझᏛޑࡌҥᐒڋǵಔԋϷࢎᄬǶӭᅿଞჹଯǵե฻෌ނಒझᏛಔԋޑ

ൂਲ਼לᡏቶݱҔܭᒣ᛽ނᅿϷځس಍Ǵٯӵଯۓက܄௖ଞǴх֖ 5 Կ 7 ঁൂᗐޑ neoglycoproteinǶ

! ! ൂਲ਼לᡏ҂ٰයఈૈᔈҔܭ୏ᄊس಍Ǵӵಒझޑ่ᄬፓ᏾ǵϸᔈғނว৖Ϸુ

ॐϐૻ৲Ǵܭϩᜪፓࢗගٮ෌ނಒझᏛϩϯว৖׳ቨᗡޑຎഁ(Knox, 1997)Ƕ

ୖ

ୖǵġ၂ᡍࢬำკ

α-amylase amyloglucosidase

Crude polysaccharides

Water-soluble nondigestible polysaccharide

Digestible polysaccharide (1,4;1,6-α-D-glucan)

Anion exchange chromatography

F1.F2.F3.F4

Immunoaffinity

l l

Size exclusion chromatography

စ

စǵġ׷਑ᆶБݤ

ಃ΋കǵġჴᡍ׷਑

ಃ΋࿯ǵġՋࢩୖኬࠔ

1.1 ٰྍ

! ! ଳᔿՋࢩୖҗ୯ϣ१ࠔϦљගٮǴаણ࿗ᐒ (RT-08Ǵٿးણ࿗ᐒǴ22000 rpm)Ǵ

຾Չϖԛણ࿗Ǵ؂ԛણ࿗࡭ុ 3 ࣾǴણ҃ᓯӸܭٛዊጃǶ

1.2 ዗Н๧ڗނϐᇙഢ

! ! ڗՋࢩୖણ҃ 50²0.1 gǴуΕ 750 mL ΒԛᇃᚖНǴ࿼ܭ 100ɗݦН੎๧ڗ 3 λਔǴаଯೲᚆЈᐒ (Beckman coulter) 8000 rpm (11325 g) ϐᙯೲᚆЈ 15 ϩដǴ ஒ๧ڗనа Whatman NO.54 ᘠરၸᘠǶ؈ᐘނа 350 mL ΒԛᇃᚖНǴܭ 100ɗݦ Н੎๧ڗ 30 ϩដǴख़ፄ๧ڗ؁ᡯǴӆஒ؈ᐘނуΕ 350 mL ΒԛᇃᚖНǴܭ 100ɗ ݦН੎๧ڗ 30 ϩដǴԏ໣Οԛ܌ளϐᘠనǴᐚᕭۓ৒Կ 1000 mLǴջࣁ዗Н๧ڗ ނ (hot- water extract)Ƕ

1.3 Нྋ܄ಉӭᗐϐᇙഢ

! ! ஒ΢ॊᇙഢϐ዗Н๧ڗނ (1000 mL)ǴуΕ 4 ७ଚᆒ؈फ़Ǵᓉ࿼႖ڹ܌ளϐӭ ᗐ؈ᐘނࣁНྋ܄ಉӭᗐ (crude polysaccharides)Ƕ

1.4 Нྋ܄ё੃ϯӭᗐϷόё੃ϯӭᗐϐᇙഢ

! ! ڗ፾ໆಉӭᗐଚᆒᝌੌనǴᚆЈѐନ΢మଚᆒǴख़ፄаଚᆒమࢱኧԛǴаΒԛ ᇃᚖНൺྋಉӭᗐ؈ᐘނǴ٠ܭ 100ɗݦН੎Πྋှ 2 λਔǴྋనհࠅࡕۓ৒ Կ 100 mLǴෳۓᅹНϯӝނ֖ໆ (ऊ 1 g)Ƕа 1 g ᅹНϯӝނࣁٯǴஒྋన pH ॶፓ

᏾Կ 6.0ǴуΕ 100 μL ऐ዗܄ α-amylaseǴܭ 95ɗН੎ύϸᔈ 30 ϩដǴհࠅԿ࠻

ྕǴஒྋన pH ॶፓ᏾Կ 7.5²0.1ǹуΕ 500 μL proteaseǴܭ 60ɗН੎ύϸᔈ 30 ϩ

ដǴհࠅԿ࠻ྕǴஒྋన pH ॶፓ᏾Կ 4.5²0.2ǹуΕ 100 μL amyloglucosidaseǴܭ 60ɗН੎ύϸᔈ 30 ϩដǴհࠅԿ࠻ྕǴۓ৒Կ 100 mLǴуΕ 4 ७ᡏᑈ (400 mL) 95%ଚᆒǴᓉ࿼႖ڹ٬ӭᗐϩη؈फ़Ƕ႖ВаᚆЈБԄڗ΢మଚᆒǴ຾Չᐚᕭѐନ ଚᆒǴࣁНྋ܄ё੃ϯӭᗐ (digestible water-soluble polysaccharides)Ƕ؈ᐘނа፾

ໆ 78%ଚᆒྋనᝌੌϩණǴమࢱ؈ᐘނ߄य़ϐλϩηǴԜమࢱ؁ᡯख़ፄ 2-3 ԛǴ؈

ᐘނջࣁНྋ܄όё੃ϯӭᗐ (water-soluble nondigestible polysaccharides)Ƕ

1.5 ഍

ڗᕴᗐໆऊ 10 mg ϐНྋ܄όё੃ϯӭᗐଚᆒᝌੌనǴᚆЈѐନ΢మଚᆒǴ ख़ፄаଚᆒమࢱኧԛǴаΒԛᇃᚖНൺྋӭᗐ؈ᐘނǴᚆЈڗள΢మϐӭᗐྋనǴ

࿶ Whatman NO.54 ᘠરၸᘠǴݙΕ഍ᚆηቫ݋ᆅࢊ (XK 26/40 Series, 300Ø26 mm i.d., GE health, Uppsala, Sweden)Ǵ༤кϐጤᡏࣁ Toyopearl DEAE-650M (Tosho, Tokyo, Japan)Ǵа 20 mM Tris མଛ 0 Mǵ0.1 Mǵ0.18 M ᆶ 0.3 M NaCl ϐࢬࢱనǴ а؂ϩដ 0.8 mL ϐࢬೲ຾ՉఊࡋؑගǴளډόӕ஥ႝ಻மࡋϐӭᗐ୔ϩǴԏ໣Ӛ

୔ϩǴ෧ᓸᐚᕭࡕа 4 ७ᡏᑈଚᆒ؇फ़ԏ໣Ӛ୔ϩϐӭᗐǶ

ಃ

ಃΒ࿯ǵġჴᡍᛰࠔᆶ၂Ꮚ 2.1 ϯᏢᛰࠔᆶ၂Ꮚ

Acetic acid, AcOH, CH3COOH ᖼԾ Merck, Darmstadt. Germany.

Acetic anhydride, AC2O, (CH3CH2)2O ᖼԾ Sigma-Aldrich, St. Louis, U.S.A.

Acetone, CH3COCH3ᖼԾ Sigma-Aldrich, St. Louis, U.S.A.

Anhydrous methanol, CH3OH (99.8%) ᖼԾ J.T.Baker, Philipsburg, NJ, U.S.A.

Barium acetate, (CH3COO)2Ba ᖼԾ Showa, Tokyo, Japan.

Bio-Rad protein assay dye concentrate ᖼԾ Bio-Rad, Hercules, CA, U,S,A.

Ethanol, C2H5OH (95%) ᖼԾ Echo Chemical, Taipei, Taiwan.

Hydrocholoric acidm HCl (37%) ᖼԾ Sigma-Aldrich, St. Louis, U.S.A.

m-hydroxydiphenyl (3-phenyl phenol)ᖼԾ Aldrich, Bothell, WA, U.S.A.

Phenol, C6H5OH ᖼԾ Wako Pure Chemical, Osaka, Japan.

Potassium chloride, KCl ᖼԾ Merck, Darmstadt. Germany.

Potassium dihydrogen phosphate, KH2PO4ᖼԾکӀપᛰ, Tokyo, Japan.

Sulfuric acid, H2SO4(95-97%)ᖼԾ Sigma-Aldrich, St. Louis, U.S.A.

Sodium azide, NaN3ᖼԾ Sigma-Aldrich, St. Louis, U.S.A.

Sodium chloride, NaCl ᖼԾ J. T. Baker, Philipsburg, NJ, U.S.A.

Sodium hydroxide, NaOH (50%, v/vǴϩ݋ભ)ᖼԾ Mallinckrodt Baker, Philipsburg, NJ, U.S.A.

Sodium nitrate, NaNO3ᖼԾ J. T. Baker, Philipsburg, NJ, U.S.A.

Sodium phosphate, NaHPO4 ᖼԾکӀપᛰ, Tokyo, Japan.

Sodium tetraborate, Na2B4O7Ǹ10H2O ᖼԾ Wako Pure Chemical, Osaka, Japan.

Trifluoroacetic acid, TFA, CF3CO2H ᖼԾ Fisher Scientific, Fair Lawn, NJ, U.S.A.

Tris (Base) ᖼԾ J. T. Baker, Philipsburg, NJ, U.S.A.

2.2 ኱

! ! L-arabinose, D-fucose, D-galactose, D-galacturonic acid, D-glucose, D-glucuronic acid, D-mannose, L-rhamnose, sorbitol, D-xylose, blue dextranǵbovine serum albumin (BSA)ǵcitrus pectinǵgum arabicǵ4-O-methyl-glucuronoxylan ฻኱ྗࠔࣣᖼԾ Sigma, St. Louis, MO, U.S.A.ǹ(1ʈ5)-α-L-arabinan ᖼԾ Megazyme, Wicklow, Ireland.ǹ pullulans ฻኱ྗࠔᖼԾ Showa Denko, Tokyo, Japan.

2.3 לᡏ

! ! LM2 (monoclonal antibody to arabinogalactan-protein)ǵLM5 (monoclonal antibody to (1ʈ4)-ß-Galactan)ǵLM6 (monoclonal antibody to (1ʈ5)-α-L-arabinan)ǵLM10 (monoclonal antibody to (1 ʈ 4)-ß-D-xylan) ǵ LM19 (monoclonal antibody to homogalacturonan) ǵ LM20 (monoclonal antibody to homogalacturonan) ǵ JIM7 (monoclonal antibody to homogalacturonan) ࣣᖼԾ Plantprobes, Leeds, UKǶ

2.4 ሇન

! ! Amyloglucosidase (3200 U/mL)ǵheat-stable α-amylase (3000 U/mL)ǵprotease (50 mg/mL)ǵglucose diagnostics (115-A) ࣣᖼԾ Sigma, St. Louis, MO, U.S.A.Ƕ

2.5 ᕗለ጗ፂన (phosphate buffer) ଛᇙ 2.5.1. ΋૓ᕗለ጗ፂనଛᇙ

! ! ᕗለ጗ፂనࣁ 0.002 M KH2PO4(0.24 g/L)ǵ0.14 M NaCl (8 g/L)ǵ0.003 M KCl (0.2 g/L)ǵ0.01M Na2HPO4(1.44 g/L)ǴpH ॶࣁ 7.4Ƕ

2.5.2. όӕ pH ॶϐᕗለ጗ፂనଛᇙ

! ! όӕ pH ॶϐᕗለ጗ፂనࣁ 0.002 M KH2PO4(0.24 g/L)ǵ0.14 M NaCl (8 g/L)ǵ