่ᄬკǶ - 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

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.,

1991)Ϸً߻૛ (Plantago major) (Samuelsen et al., 1996b)Ƕ݀ጤதـ่ᄬࣁ α-(1→4)-linked galacturonan Ǵ Ϸ rhamnogalacturonan ࣁ ମ ༸ Ǵ Ѝ ᜘ ࣁ ύ ܄ ᑗ ӵ arabinogalactanǵarabinanǵgalacto-oligosaccharides ᄬԋޑӭЛ୔ (ramified region) (Dey and Brinson, 1987)Ƕלံᡏࢲ܄ޑ݀ጤӭᗐǴڀԖ ß-3,6-galactan(Yamada et al., 1985b; Yamada et al., 1986; Samuelsen et al., 1996b)Ǵа α-L-arabinofuranosidase բҔ ࡕǴ٠ό཮ׯᡂ arabino-ß-3,6-galactan ޑࢲ܄Ǵᇥܴ (1→3,6)-ß-galactan ࣁלံᡏ ࢲ܄ϐख़ा่ᄬ(Yamada et al., 1987b; Kiyohara et al., 1988; 1989b; Yamada et al., 1989)Ƕ(1→3,6)-ß-galactan ёᆶ ß-glucosyl-Yariv antigen ᒱӝౢғआලՅ؈ᐘ(Clarke

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

(Kiyohara et al., 1989c)Ǵ౽ନ AGIIb-1 ύ arabinan Ϸ arabinogalactan ޑ arabinose ϩ ЍǴёቚуࢲϯံᡏ alternative pathwayǴᡉҢ arabinose ϩЍ׭ڋ AGIIb-1 ࢲ܄

(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-I)(Kiyohara et al., 1987)ǴӆҔ arabinofuranosidase Ϸ exo-ß-D-(1→3)-galactanase ೀ

౛ N-I ࡕϝڀԖלံᡏࢲ܄Ƕஒ N-I аጤᡏቫ݋ࡕளύϩηໆӭᗐǴࣁ GN-1A Ϸ G1BǴࣣڀԖံᡏࢲϯ੝܄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

੮Ǵ߄Ңࢲ܄ӭᗐё೏֎ԏǵ੃ϯǴӧط᠌຾ՉжᖴǴԶځдӭᗐ߾ӧᆶڙᡏ่ӝ ࡕڈᐟӄي܈ဉၰޑխࣝಒझ(Czop et al., 1990)Ƕ

ಃ

ಃ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

ڰۓ࣬

! ! 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

^̛= Vo+

̇

^̚

V

iǴViࣁЍ࡭ϾࢰޑᡏᑈǹVoࣁคᅉ੮ؑගᡏᑈϐݢঢ়ǴջࣁϾࢰѦޑ ᡏᑈǶϩଛ߯ኧ (distribution coefficient)

̇

^̚ёჹᔈ ViޑϩѲܭϩηǶਥᏵۓကǴ

΋ঁֹӄ௨ନϩηϐϩଛ߯ኧࣁ႟ǴԶ྽΋ϩη೯ၸ܌ԖϾࢰਔǴ

̇

^̚=1ǶV^̛ǵ Voǵ

V

i ᆶ Vtϐᜢ߯ёـკΜǴ؂΋ϩηޑࢬࢱ౜ຝࣣёа V^̛܈

̇

^̚߄ҢǴӢ V^̛ڗ،ܭ

ᆅࢊЁκǴࡺϩଛ߯ኧ (

̇

^̚) ၨࣁදၹҔаඔॊނ፦ޑϩᚆ੝܄Ƕ

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

ڰۓ࣬

! ! ΋૓٬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

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

ൌ

ሾሿሾሿ^ି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

БԄ

! ! 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 ൂ

ൂਲ਼לᡏ (anti-(1ʈ5)-α-L-arabinan monoclonal antibody)

! ! 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 ൂ

ൂਲ਼לᡏ (anti-homogalacturonan monoclonal antibody)

! ! 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

ᅿηᗹన (seed mucilage) ଺ࣁלচǴམଛ٢Ꮚ (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 LM20(Verhertbruggen et al., 2009; Marcus et al., 2010)Ƕ

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 ൂ

ൂਲ਼לᡏᆶ෌ނಒझᏛӭᗐᒃکϸᔈϐᔈҔ

! ! 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.,

1990; Knox et al., 1991)Ǵҗ Anti-AGP ൂਲ਼לᡏᡉҢځ߄౜ቶݱޑวػፓ௓ᐒڋǶ

ൂ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

Ϸ arabinogalactan ϐ੝ۓלՈమ(Northcote et al., 1989)ǶӭኧՏܭ෌ނಒझᏛ΢ޑ

ೈ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 ഍

഍ᚆηҬඤᐋિቫ݋ϐНྋ܄όё੃ϯӭᗐ୔ϩ

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 ኱

኱ྗࠔ

! ! 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

0.003 M KCl (0.2g/L)ǵ0.01M Na2HPO4(1.44 g/L)Ǵа HCl ᆶ NaOH ፓ᏾ځ pH ॶࣁ 3.4ǵ4.4ǵ5.4ǵ6.4ǵ7.4ǵ8.4ǵ9.4Ƕ

2.5.3. ό

όӕ NaCl ᐚࡋϐᕗለ጗ፂనଛᇙ

! ! όӕ NaCl ᐚࡋϐᕗለ጗ፂనࣁ 0.002 M KH2PO4(0.24 g/L)ǵ0.003 M KCl (0.2 g/L)ǵ0.01 M Na2HPO4(1.44 g/L)ǴуΕόӕ NaCl ֖ໆаଛᇙόӕ NaCl ᐚࡋǴ0 M (҂బу)ǵ0.14 M NaCl (8.19 g/L)ǵ0.4 M NaCl (23.4 g/L)ǵ0.8 M NaCl (48.6 g/L)ǵ 1.6 M NaCl (93.6 g/L)ǵ3.2 M NaCl (187.2 g/L)ǵ4 M NaCl (234 g/L)ǴpH ॶࣁ 7.4Ƕ

ಃΟ࿯ǵġሺᏔ೛ഢ

ELISA reader, sunrise ᖼԾ Tecan Trading AG, Switzerland.

ᚆЈᐒ,ᖼԾ Beckman coulter, California, U.S.A

ᚆЈᐒ, Kubota 5100 ᖼԾᚈᡳҾ཰Ԗज़ϦљǴཥчѱǴѠ᡼

ϩࢤԏ໣Ꮤ, Frac 920ǵRediFrac ࣣᖼԾ GE health, Uppsala, Sweden.

pH meter, PC-310 ᖼԾ SuntexǴ΢਱ࣽמԖज़ϦљǴѠࠄѱǴѠ᡼

ٛዊጃ, DX206 ᖼԾѠ᡼ٛዊࣽמިҽԖज़Ϧљ (ԏᙒৎ)ǴѠчѱǴѠ᡼

᎜ᕏН੎ኲ, Power sonic 420 ᖼԾ΢ੀሺᏔިҽԖज़ϦљǴཥчѱǴѠ᡼

Нኲᠳ܏Ꮤ, MSW-20 ᖼԾ୯ԋࣽᏢሺᏔϦљǴཥчѱǴѠ᡼

ϩӀӀࡋी, Helio Omegasp spectrophotometer ᖼԾ Thermo Fisher Scientific Inc., Walthan, U.S.A.

෧ᓸᐚᕭᐒ, RE-111 ᖼԾ Buchi, Flawil, Switzerland.

НࢬԄܜ਻س಍, EYELA A1000S ᖼԾۘ଻ިҽԖज़ϦљǴѠчѱǴѠ᡼

ಃ

ಃѤ࿯ǵġϯᏢϩ݋Бݤ

4.1 ᕴᗐ֖ໆෳۓ (Total carbohydrate determination)

! ! ٩Ᏽ Dubois ฻Γ (1956) ϐෳۓБݤ (phenol-sulfuric acid method)Ǵа 10ǵ30ǵ 50ǵ70ǵ90 μg/mL ϐ D-glucose բࣁ኱ྗԔጕኬࠔǶڗ 200 μL ኱ྗࠔϷࡑෳኬࠔ ྋనуΕ 200 μL 5% (w/v) phenol ྋనࡕа 1 mL ᐚ౷ለ (18 M H2SO4) ࠟޔؑΕǴ

٬ϐϸᔈևՅǴᓉ࿼ 10 ϩដǴ᎜ᕏషӝ֡Ϭࡕӆᓉ࿼ 30 ϩដǴෳۓ OD 490 nm

֎ԏॶ(Dubois et al., 1956)Ƕ

4.2 α-D-glucose ֖ໆෳۓ (α-D-glucose determination)

! ! ୖԵ Karkalas ӧ 1985 ԃගрϐဟ๻ᑗ֖ໆෳۓݤ (GOD-POD)Ǵଛᇙဟ๻ᑗ

኱ྗྋన 10ǵ30ǵ50ǵ70ǵ90 ug/mL ᇙբ኱ྗԔጕǶڗဟ๻ᑗ኱ྗྋనᆶኬࠔӚ 1 mL уΕᔠෳ၂Ꮚ 10 uLǴ࠻ྕϸᔈ 15 ϩដǴෳۓ OD 505 nm ֎Ӏॶ(Karkalas, 1985)Ƕᔠෳ၂Ꮚύ֖Ԗ glucose oxida 20000 u/Lǵperoxidase 1200 u/L ᆶ 4-AAP 0.246 mmol/LǶ

4.3 ⾺ᑗለ֖ໆෳۓ (Uronic acid determination)

! ! ୖԵ Blumenkrantz ک Asboe ӧ 1973 ԃගрϐෳۓБݤ (m-hydroxydiphenyl method)Ƕ10ǵ30ǵ50ǵ70ǵ90 μg/mL ϐ D-galacturonic acid բࣁ኱ྗԔጕኬࠔǶڗ 200 μL ኱ྗࠔϷࡑෳኬࠔྋనܭӇ੎ϣуΕ 1.2 mL H2SO4/tetraborate ྋనǴܭӇ

੎ϣᓉ࿼ 5 ϩដࡕ᎜ᕏషӝ֡ϬǴ࿼Ε 100ʚݦН੎ύу዗ 5 ϩដǴу዗ࡕ࿼ܭӇ

੎ύ 5 ϩដࡑځհࠅǴуΕ 20 μL m-hydroxydiphenyl ၂ᏊǴ᎜ᕏషӝ֡Ϭࡕ 15 ϩ ដෳۓ OD 520 nm ֎ԏॶǶӧݦН੎у዗ࡕϸᔈྋనऩևསआՅ܈ፃՅǴ߾ሡᇙ ഢޜқኬࠔ (blank sample)Ǵޜқኬࠔෳۓࢬำᆶ኱ྗኬࠔ࣬ӕǴ୤٬Ҕ 0.5% NaOH ڗж m-hydroxydiphenyl ၂ᏊǶஒኬࠔϐ OD 520 nm ֎ԏॶԌନޜқኬࠔϐ OD 520 nm ֎ԏॶࡕӆ٩኱ྗԔጕीᆉኬࠔ⾺ᗐለᐚࡋ(Blumenkrantz and Asboe-Hansen,

1973)Ƕ

4.4 ೈ

ೈқ፦֖ໆෳۓ (Protein determination)

! ! ୖԵ Bradford ӧ 1976 ԃගрϐೈқ፦-ࢉ਑่ӝෳۓБݤ (Dye-binding assay)Ǵଛᇙ 2ǵ4ǵ6ǵ8ǵ10 μg/mL ϐФՈమೈқ (bovine serum albumin, BSA) բ ࣁ኱ྗԔጕኬࠔǶஒ Bio-Rad ၂Ꮚ (Bio-Rad protein assay dye concentrate) аΒԛ ᇃᚖНีញ 5 ७Ǵڗ 50 μL ݙΕ 96 ϾዬύǴуΕ 200 μL ኱ྗࠔϷࡑෳኬࠔྋనǴ ܭ࠻ྕΠᓉ࿼ 5 ϩដࡕа ELISA reader ෳۓ OS 650 nm ֎ԏॶ(Bradford, 1976)Ƕ

4.5 KDO ֖ໆෳۓ (KDO determination)

! ! ٩Ᏽ Karkhanis ᆶ York ฻ΓϐБݤ٠уаঅׯǴଛᇙ KDO 4-20 μg/mL ଺ࣁ኱

ྗԔጕǶڗ 400 μL ޑ኱ྗࠔϷኬࠔྋనǴуΕ 100 μL 1N H2SO4Ǵܭ 100ʚݦН੎

ύϸᔈ 30 ϩដǴհࠅࡕᚆЈѐନόྋނǴуΕ 250 μL 0.04 M HIO4 (ྋܭ 0.125 N H2SO4)Ǵ֡Ϭ᎜ᕏషӝǴܭ࠻ྕΠܫ࿼ 20 ϩដǴуΕ 250 μL 2% Na2SO3(ྋܭ 0.5 N HCl)Ǵ֡Ϭ᎜ᕏషӝޔډලՅ੃ѨǴӆуΕ 500 μL 0.6% TBA (thiobarbituric acid)Ǵ

֡Ϭ᎜ᕏషӝࡕܭ 100ʚݦН੎ύϸᔈ 15 ϩដǴອ዗уΕ 1 mL DMSOǴհࠅԿ࠻

ྕǴෳۓ OD 548 nmǶ

4.6 ሇનೱ่խࣝ֎ߕݤ (enzyme-linked immunosorbent assay, ELISA)

! ! ୖԵ Verhoef ฻ΓϐБݤǴஒኬࠔྋన (לচ) а pH 7.4 ϐᕗለ጗ፂన (؂Ϧ ϲ֖ 0.2 g KClǵ0.24 g KH2PO4ǵ8 g NaCl ک 1.44 g Na2HPO4) ຾Չ፾྽ีញǴஒี

ញϐኬࠔ 100 μL уΕ 96 Ͼዬύ (Nunc 442404, F96 Maxisorp, Thermo Fisher Scientific Inc., Roskilde, Denmark)Ǵܭ 4ɗᓉ࿼႖ڹ (εܭ 16 λਔ) ຾Չלচ֎ߕ ϸᔈ (coating)Ƕ֎ߕϸᔈࡕॹନኬࠔྋనǴаΒԛᇃᚖНዎࢱ 4 ԛ (300 μL/well)Ǵ ӆа 3% (w/v) ಥિѪણ (nonfat bovine milk powder, Sigma) ϐᕗለ጗ፂన (200

μL/well) ຾Չ༤༞ϸᔈ (blocking)Ǵܭ࠻ྕᓉ࿼ 1 λਔǶॹନዬϣྋనǴख़ፄዎࢱ

؁ᡯ 4 ԛǴуΕ 100 μL ΋ભלᡏ (LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20ǵ JIM7 ࣣаᕗለ጗ፂనีញ 20 ७)Ǵܭ࠻ྕϸᔈ 2.5 λਔǴॹନࡕख़ፄዎࢱ؁ᡯ 6 ԛǶуΕ 100 μL ೱ่ᇺਥၸ਼ϯނሇનޑΒભלᡏ (goat anti-rat IgG coupled to horseradish peroxidase, HRPǴа 1% (w/v) ಥિѪણᕗለ጗ፂనีញ 2000 ७)Ǵܭ

࠻ྕΠϸᔈ 1.5 λਔǴॹନࡕख़ፄዎࢱ؁ᡯ 6 ԛǴуΕ 150 μL ϸᔈڙ፦ 3,3’,5,5’-tetramethylbenzidine (TMB) (аΒԛᇃᚖНีញ 2 ७)Ǵ࠻ྕΠᗉӀ 10 ϩដ຾Չև ՅϸᔈǴуΕ 50 μL 1M H2SO4ಖЗϸᔈǴа ELISA reader ෳۓ OD 450 nm ֎ԏॶ (Verhoef et al., 2009)Ƕ

4.7 ൂ

ൂᗐಔԋϩ݋

! ! ຾Չൂᗐಔԋϩ݋ϐ߻ǴሡӃа⾺ᑗለ֖ໆෳۓຑ՗⾺ᑗለϐ֖ໆǴऩኬࠔύ

֖Ԗለ܄ᑗǴ߾ᎃ߈ޑᑗ㧿ᗖܭለ܄ᕉნύᛙۓǴคݤܭΟࢧᎉለ (trifluoroacetic acid, TFA) ᕉნύНှֹӄǴԶ⾺ᑗለൂᡏ೏НှࡕǴܰफ़ှԋߚᅹНϯӝނϐౢ

ނǴόճܭϩ݋ǴӢԜǴሡा೸ၸ methanolysis ؁ᡯගଯ֖Ԗ⾺ᑗለኬࠔޑНှ౗Ǵ

֡٬ϐࣁ஥ԖҘ୷ޑᑗ㧿 (methyl glycosides)Ǵӆа TFA ஒҘ୷НှǴளډֹ᏾ޑ

⾺ᑗለൂᡏǶ

4.7.1. ࡑෳኬࠔ߻ೀ౛

! ! ڗᕴᗐໆऊ 1 mg ޑࡑෳኬࠔྋన࿼ܭኬࠔ౟ύǴ٩ׇуΕ 95% Όᎇǵ99%Ό ᎇϷคНҘᎇ෧ᓸᐚᕭǴ٬ኬࠔଳᔿǴӆаϿໆคНҘᎇஒኬࠔ࿼ΕНှᆅύǴ٠ а੿ޜܜ਻ѐନคНҘᎇǶ

4.7.2. Οࢧᎉለ (trifluoroacetic acid, TFA) Нှ

! ! уΕ 0.5 mL 4 M TFA Нྋన (ەܭჴᡍ྽Вଛᇙ) ԿНှᆅύǴஒᕉნܜԋ

੿ޜǴа 100ɗଳ੎ϸᔈ 3 λਔǴ෧ᓸᐚᕭѐନ TFA НྋనԿᕉნࣁύ܄ (аቶҔ ၂રෳۓ pH ॶ=7)Ǵа 1 mL ຬપНൺྋНှౢނǴ࿶ 0.45 μm nylon ᘠጢ (Nylon, 0.45 μm, Aglela Technologies) ၸᘠǴа HPAEC-PAD س಍ϩ݋Ǵϩ݋ύ܄ᗐచҹࣁ 19 mM NaOH (֖ 1 mM barium acetate) НྋనǴࢬೲࣁ 0.5 mL/minǴКჹ L-arabinoseǵ L-fucoseǵD-galactoseǵD-glucoseǵD-mannoseǵL-rhamnoseǵD-xylose ฻኱ྗࠔޑ ᅉ੮ਔ໔ǵճҔӚݢঢ়ޑᑈϩय़ᑈीᆉрൂᗐವԸಔԋ (mol%)Ƕ

4.7.3.Ҙ

Ҙ✊ϯϸᔈ (Methanolysis)

! ! ਥᏵ Deruiter (1992)ǵBertaud (2002) Ϸ Talaga (2002)฻ΓϐୖԵБݤǴڗᕴᗐ ໆऊ 1 mg ޑࡑෳኬࠔྋన࿼ܭ੿ޜНှᆅύǴ෧ᓸᐚᕭԿଳǴуΕ 1 mL 2 M HCl/MeOHǴଛᇙБݤࣁǴܭӇ੎ύஒ 1.41 mL acetyl chloride ጗ᄌуΕคНҘᎇ ύǴ٠аคНҘᎇۓ৒Կ 10 mLǶஒᕉნܜԋ੿ޜࡕǴܭ 80ɗଳ੎ϸᔈ 12 λਔǴ ෧ᓸᐚᕭѐନคНҘᎇǴуΕ 1 mL 2 M TFAǴஒᕉნܜԋ੿ޜǴܭ 100ɗଳ੎ϸ ᔈ 1 λਔǴ෧ᓸᐚᕭѐନ TFA НྋనԿᕉნࣁύ܄ (аቶҔ၂રෳۓ pH ॶ=7)Ǵ а 1mL ຬપНൺྋНှౢނǴ࿶ 0.45 μm nylon ᘠጢၸᘠǴа HPAEC-PAD س಍ϩ

݋Ǵϩ݋ύ܄ᗐచҹӕ 4.5.2ǹለ܄ᗐϩ݋చҹࣁ 75 mM NaOH (֖ 1 mM barium acetate ک 150 mM sodium acetate) НྋనǴࢬೲࣁ 1 mL/minǴКჹ D-galacturonic acid Ϸ D-glucuronic acid ฻኱ྗࠔޑᅉ੮ਔ໔ǵճҔӚݢঢ়ޑᑈϩय़ᑈीᆉрൂᗐ ವԸಔԋ (mol%)(Deruiter et al., 1992; Bertaud et al., 2002; Talaga et al., 2002)Ƕ

4.7.4. ϩ݋س಍

! ! ଯਏૈ഍ᚆηҬඤቫ݋س಍ (high performance anion exchange chromatography with PAD,HPAEC-PAD ᖼԾ Jasco, Ino., Japan.)ǵଯਏૈ഍ᚆηҬඤቫ݋ߥៈᆅࢊǴ CarboPac PA1 guard column (2% polystyrene cross-linked with divinylbenzene, 4 mm i.d.Ø5 cm, 10 μm) Ϸϩ݋ᆅࢊǴCarboPac PA1 (2% polystyrene cross-linked with

divinylbenzene, 4 mm i.d.Ø5 cm, 10 μm) ֡ᖼԾ Dionex, Sunnyvale, CAǶ

4.8 ϩ

ϩηໆෳۓ

4.8.1. ࡑෳኬࠔ߻ೀ౛

! ! ڗᕴᗐໆऊ 1 mg ϐжෳኬࠔྋనǴ࿶ 1.2 μm PVDF ᘠጢ (PVDF, Dia.25 mm, Critical)ၸᘠഢҔǶ

4.8.2. ኱ྗࠔ

! ! ଛᇙ 1 mg/mL dextran (2Ø10⁶Da)ǵglucose ᆶ pullulans ኱ྗࠔྋన (P5, 5.9Ø10³ DaǹP10, 0.96Ø10⁴ DaǹP20, 2.11Ø10⁴ DaǹP50, 4.71Ø10⁴ DaǹP100, 1.07Ø10⁵ Daǹ P200, 2.00Ø10⁵DaǹP400, 3.75Ø10⁵DaǹP800, 7.08Ø10⁵Da)Ǵ࿶ 0.45 μm nylon ᘠጢ ၸᘠഢҔǶ

4.8.3. ϩ݋س಍

! ! ଯ ਏ ૈ ϩ η ᑔ ቫ ݋ س ಍ (high performance size exclusion chromatography, HPSEC)Ǵᔅ੅ၮଌس಍ࣁ PU-980 Plus quatermary-gradient pump (Jasco, Inc., Japan)ǹ ݙΕᡏᑈ 200 μLǴݙΕᏔࣁ 7215 Cotati (California, U.S.A.)ǹୀෳᏔࣁש৔౗ෳۓ Ꮤ, Shodex, RI-71 refractive index detector (Showa Denko, Tokyo, Japan)ǹߥៈᆅࢊ

TSK guard column PWH (7.5 mm i.d.Ø7.5 cm,12 μm)Ϸϩ݋ᆅࢊǴTSK 4000 PWXL column (7.5 mm i.d.Ø30 cm,17 μm) Սᖄ TSK 3000 PWXL column (7.5 mm i.d.Ø30 cm,17 μm) (Tosoh, Tokyo, Japan.)Ǵᆅࢊྕࡋࣁ 80ɗǴၗ਑ϩ݋س಍ࣁ SISC32 (ૻ

๮ިҽԖज़ϦљǴѠчѱǴѠ᡼)Ƕࢬࢱచҹࣁ 0.3 M NaNO3(֖ 0.02% NaN3)Ǵࢬ ೲ 0.5 mL/minǶ

4.9 HPSEC Ս

Սᖄ ELISA assay

! ! ڗᕴᗐໆऊ 1 mg ϐжෳኬࠔྋనǴ࿶ 1.2 μm PVDF ᘠጢ (PVDF, Dia.25 mm, Critical) ၸᘠࡕ࿶ HPSEC ϩ݋ࡕаϩࢤԏ໣Ꮤ (RediFrac ᖼԾ GE health, Uppsala, Sweden.) а؂ᆅ 10 ᅀ (0.29 mL-0.31 mL) ຾Չԏ໣Ǵаচన܈ีញ 100 ७ࡕݙܭ 96 ϾዬǴ຾Չ ELISA ϩ݋ǶࣁΑஒ ELISA ϩ݋კᆶ HPSEC ϐቫ݋კ຾ՉӝٳǴ

໪ዴᇡ ELISA ϩ݋კϐ distribition coefficient (DC)ǶዴᇡϐБݤࣁݙΕ 10 mg/mL ϐ blue dextranǴаϩࢤԏ໣Ꮤࡪྣ΢ॊБݤԏ໣Ǵෳۓ OD 630 nm ϐ֎ӀॶǴջ ёளޕ blue dextran ໒ۈࢬрϐᆅኧǴ४΢؂ᆅ܌ԏϐᡏᑈǴջࣁ VoǶVt ߾аᑗࡋ

ीෳໆځש৔౗Ǵа HPSEC ࢬࢱనᘜ႟ǴёаෳளࢬࢱనᆶНঢ়ϐ໔࣬౦ޑש৔

ॶǴڗНঢ়߻΋ᆅǴ४΢؂ᆅ܌ԏϐᡏᑈǴջࣁ VtǴҗԜ Vo ᆶ Vt ॶջёෳளϩࢤ ԏ໣Ꮤϐ DC ॶǴ٠ёаᆶ HPSEC ϐ RI კ᛼ӝٳǶ

ಃϖ࿯ǵġ಍ीϩ݋

! ! ჴᡍኧᏵ่݀֡аѳ֡ॶ²኱ྗৡ߄ҢǴኧᏵϩ݋а SAS 9.3 ঺း೬ᡏ аᎅМཥӭᡂୱෳᡍݤ (Duncan’s new multiple range test) ຾ՉᔠۓǴP ॶ

<0.05 ຎࣁԖᡉ๱ৡ౦Ƕ

Ҵ

Ҵǵġ่݀ᆶ૸ፕ

ಃ΋കǵġНྋ܄όё੃ϯӭᗐ

ಃ΋࿯ġǵНྋ܄όё੃ϯӭᗐ୔ϩϐಔԋϩ݋

! ! ՋࢩୖϐН๧నࣁԜᅿ१׷ޑЬाឪ१БԄǴН๧ނКٯࣁচ਑ϐ 58.2%Ǵಉӭᗐࣁ 25.04%ǴԜಉӭᗐ֖Ԗೈқ፦ 1.18%Ǵஒಉӭᗐ຾Չ࿚నࢉՅǴ և౜ᙔ๋Յё௢ፕՋࢩୖಉӭᗐύ֖ԖεໆޑᐘણǶࣁΑᕕှΓୖ१׷܌ឪΕޑ ӭᗐ܄፦Ǵҁჴᡍஒ ಉӭ ᗐа protease բҔѐନೈқ፦ࡕǴ ᆶ α-amylaseǵ amyloglucosidase ຾ՉբҔǴёஒಉӭᗐϩࣁё੃ϯӭᗐ (digestible polysaccharide) ᆶНྋ܄όё੃ϯӭᗐ (water-soluble nondigestible polysaccharide)Ƕё੃ϯӭᑗ࿶

җဟ๻ᑗ਼ϯ䁙-ၸ਼ϯނ䁙 (glucose oxidase-peroxidase, GOD-POD) ෳۓǴ൤֖

glucoseǴ௢ෳࢂҗ α-amylase ᆶ amyloglucosidase ܌Нှޑᐘણ่ᄬǴ(1,4;1,6)-α-D-glucanǶ࿶ሇનНှࡕ܌ளϐНྋ܄όё੃ϯӭᗐǴᆶಉӭᗐ࣬КǴځൂᗐಔԋ ύ glucose ֖ໆεࣁ෧ϿǴҗ 93.86%फ़Կ 5.19%Ƕё੃ϯӭᗐǴЬाࣁᐘણ܌ᄬ ԋǴࣁ (1,4;1,6)-α-D-glucanǴэಉӭᗐϐ 95.7%ǴНྋ܄όё੃ϯӭᗐ߾э 4.3%

(݅, 2012)ǶஒНྋ܄όё੃ϯӭᗐа഍ᚆηҬඤቫ݋ᆅࢊϩ݋Ǵܭ 20 mM Tris ֖ NaCl ᐚࡋ 0ǵ0.1ǵ0.18ǵ0.3 M ϐࢬࢱనؑගǴёளѤঁ୔ϩ F1ǵF2ǵF3 ᆶ F4 (კ ΜѤ)Ƕ഍ᚆηҬඤᐋિϩᚆБݤࣁ٩Ᏽځႝ಻܄ǴF1 ୔ϩҗᡶᐚࡋࣁ 0 M ܌ؑග р౜Ǵ⾺ᗐለ֖ໆե (߄΋)Ǵаύ܄ᑗ arabinose (18.92%) ᆶ galactose (68.53%)ࣁ Ь (߄Β)ǶF2 ࣁᡶᐚࡋ 0.1 M ܌р౜Ǵಔԋϝа arabinose (47.35%) ᆶ galactose (40.71%) ࣁЬǴ௢ෳԜΒ୔ϩࣁ݀ጤ΢ RG-I ่ᄬϐϩЍ AGIǶᒿࢬࢱనᡶᐚࡋຫ ଯǴࢬࢱрޑӭᗐ஥Ԗႝ಻܄ຫଯǴᡶᐚࡋ 0.18 M ࢬࢱрϐ F3 ୔ϩǴځ⾺ᑗለ֖

ໆэᕴᗐໆϐ 35.30 %ǶF4 ࣁᡶᐚࡋ 0.3 M ܌ࢬࢱрٰޑ୔ϩǴа galacturonic acid ࣁЬाಔԋǴ⾺ᗐለэᕴᗐໆ 197.13%ǴচӢࣁ F4 ࢂҗଯໆޑъ٢ᑗ⾺ለ܌ಔԋǴ ՠࢂаᕴᗐໆޑෳۓݤǴለ܄ᑗคݤ೏НှևՅǴ཮ե՗ F4 ϐᕴᗐ֖ໆǶF3 ᆶ F4

ࣣ֖Ԗ glucuronic acidǴࡺࣁ RG-I ΢ϐ AGII ϩЍǶ

߄΋ǵНྋ܄όё੃ϯӭᗐ࿶җ DEAE ቫ݋܌ϩᚆޑѤᅿϩቫӭᗐϐ୷ҁಔԋǶ Table 1. Chemical compositions of four fractions from water-soluble nondigestible polysaccharides separated by DEAE chromatography.

Fraction %

Protein /Carbohydrate ratio Uronic acid in carbohydrate

Protein

:Carbohydrate

(%)

F1 18.51±6.68 1:9.11 1.36±0.01

F2 19.90±3.03 1:9.14 1.04±0.02

F3 28.28±2.67 1:6.83 35.30±0.07

F4 33.31±6.61 1:3.76 197.13±1.55

aOn basis of carbohydrate contents of four fractions.

bValues from Coomassie blue method (Bio-red protein assay reagent ) using BSA as standard.

cValues from phenol-sulfuric acid method using glucose as standard.

dOn carbohydrate basis and the uronic acid values from m-hydroxydiphenyl method using galacturonic acid as standard.

eMeans²standard deviation, n=3.

߄ΒǵНྋ܄όё੃ϯӭᗐ࿶җ DEAE ቫ݋܌ϩᚆϐѤঁ୔ϩӭᗐځӚձൂᗐಔ ԋ (݅, 2012)Ƕ

Table 2. Monosaccharide molar compositions of four fractions from water-soluble nondigestible polysaccharides separated by DEAE chromatography.

Fraction Molar ratio of sugar composition (%)

Ara Gal Glc Man Rha GalA GlcA

F1 18.92 68.53 5.41 7.14 ND ND ND

F2 47.35 40.71 4.55 7.39 ND ND ND

F3 55.18 30.72 ND ND 4.23 6.33 3.55

F4 21.31 17.71 7.96 ND 9.16 43.54 0.3

კΜѤǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩ݋კǶ

Figure 14. Elution profile of water-soluble nondigestible polysaccharides of P.

quinquefolius on DEAE chromatography, eluted with 20mM Tris and a stepwise

gradient of NaCl.

ಃ

ಃΒ࿯ġǵНྋ܄όё੃ϯӭᗐύӚӭᗐ୔ϩϐϩηໆ

! ! ࣁᕕှӭᗐϐϩѲǴ٬Ҕ HPSEC ຾Չϩηໆϩ݋ǶӭᗐڀԖፄᚇޑ่ᄬǴค

ൂ΋ϩηໆǴϩηঁኧό཮ֹӄ࣬ӕǴѸ໪аኧໆѳ֡ϩηໆ (number average molecular weight, Mn) Ϸख़ໆѳ֡ϩηໆ (weight average molecular wight, Mw) ٰ ߄ҢǴځύΞаख़ໆѳ֡ϩηໆၨૈж߄ӭᗐޑ੝܄ǴٿޣޑКॶࣁ polydispersity index, PI (Mw/Mn) ߾߄ҢϩηໆޑϩѲ௃׎Ƕ

! ! ஒՋࢩୖНྋ܄όё੃ϯӭᗐа഍ᚆηҬඤᐋિϩᚆϐ F1ǵF2ǵF3 ᆶ F4 ୔ ϩೀ౛ RAW 264.7 ႵѮᏘಒझǴว౜ F3 ᆶ F4 ୔ϩӧᐚࡋ 100 μg/mL а΢ᡉ๱ڈ ᐟ TNF-α ᆶ NO ғԋ(݅,2012)Ƕ຾΋؁ϩ݋ F1-F4 ୔ϩϐϩηໆ (კΜϖ-კΜΖ)Ǵ F1ǵF2 ୔ϩځ Mw ࣁ 71ǵ115 kDaǴPI ࣁ 3.62 ᆶ 2.01ǹF3 ᆶ F4 ࣁ 273ǵ172 kDaǴ PI ࣁ 3.87 ᆶ 2.94 (߄Ο)ǶڀԖڈᐟಒझᐟનғԋϐ F3 ᆶ F4 ୔ϩځϩηໆϷ PI ࣣ

εܭ F1 ᆶ F2ǴԜ่݀ёჹᔈܭ 2002 ԃ Shin ฻Γஒ๧ڗϐΓୖӭᗐ (ϩηໆ 150 kDa) ڈᐟѮᏘಒझǴӧᐚࡋ 100 μg/mL а΢ܴᡉڈᐟ TNF-α Ϸ NOǴܭ 10 ug/mL а΢ڈᐟ IL-1βǵIL-6 ᆶ IFN-γ ϐғԋ(Shin et al., 2002)ǶKabat ᆶ Bezer ࡰрǴጋ ᆒޑϩηໆεܭ 90 kDa ჹΓᜪڀԖխࣝלচ܄ǴԶ྽ϩηໆλܭ 50 kDa ਔ߾όڀ խࣝڈᐟ܄(Kabat and Bezer, 1958)Ƕ1999 ԃ Yamada ᆶ Kiyohara(Yamada and Kiyohara, 1999) ගрύ૛ᛰύڀԖࢲϯံᡏࢲ܄ϐӭᗐǴъኧа΢ځϩηໆεܭ 128 kDaǴ߄ҢӭᗐϐϩηໆελϷ PI ॶࣁ،ۓځڈᐟಒझᐟનᆶࢂցڀԖխࣝࢲ

܄ϐᜢᗖӢનǶ

კΜϖǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F1 ϩηໆკ᛼Ƕ Figure 15. HPSEC elution profiles of fraction one of water-soluble nondigestible polysaccharides of P. quinquefolius on DEAE chromatography.

კΜϤǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F2 ϩηໆკ᛼Ƕ Figure 16. HPSEC elution profiles of fraction two of water-soluble nondigestible polysaccharides of P. quinquefolius on DEAE chromatography.

კΜΎǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F3 ϩηໆკ᛼Ƕ Figure 17. HPSEC elution profiles of fraction three of water-soluble nondigestible polysaccharides of P. quinquefolius on DEAE chromatography.

კΜΖǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F4 ϩηໆკ᛼Ƕ Figure 18. HPSEC elution profiles of fraction four of water-soluble nondigestible polysaccharides of P. quinquefolius on DEAE chromatography.

߄ΟǵНྋ܄όё੃ϯӭᗐ࿶җ DEAE ቫ݋܌ϩᚆϐѤᅿ୔ϩӭᗐځӚձϩη ໆǶ

Table 3. The molecule weight of four fractions water-soluble nondigestible polysaccharides of P. quinquefolius on DEAE chromatography^a

Fraction Molecular weight of fraction (ൈ ૚૙

^૞

)

Mn Mw PI

F1 0.20±0.00 0.71±0.01 3.62±0.08

F2 0.57±0.00 1.15±0.01 2.01±0.01

F3 0.70±0.03 2.73±0.20 3.87±0.11

F4 0.58±0.00 1.72±0.00 2.94±0.02

aThe value was determined by HPSEC with pullulans as standards.

bMn: number average weight (kDa); Mw: weight average weight; PI: polydispersity index (Mw/Mn).

cMeans²standard deviation, n=3.

ಃ

ಃΒകǵġሇનೱ่խࣝ֎ߕϩ݋ݤന፾ϯచҹϩ݋

! ! ሇનೱ่խࣝ֎ߕϩ݋ݤ (enzyme linked immunosorbent assay, ELISA)Ǵࣁ ᙖҗלᡏ-לচ໔ڀ஑΋܄ޑᒃکΚǴ຾Չϩ݋᠘ۓǶҔܭڰۓלচ܈לᡏǴ٬Ҕ ڀԖமᗖ่Κޑ൳ᅿ༟਑׷፦ӵᆫशΌ౎ (polystyrene)(Leininger et al., 1966; Catt and Tregear, 1967)ǴҞ߻ჹܭځᗖ่БԄۘ҂ܴᕕǴ௢ෳࢂҗ౧НϷᒃНΚᆶϐ࣬

ϕբҔ٬ځၲ֎ߕૈΚ(Dillman and Miller, 1972; Shirahama and Suzawa, 1985)Ƕ

ಃ΋࿯ġǵCoating buffer ϐ pH ॶᆶᚆηமࡋჹלচ֎ߕϸᔈϐቹៜ

! ! לচᙖҗ౧НբҔǵᓉႝЇΚϷΥቺґᅟΚڰۓܭ 96 Ͼዬ΢ǴࣁளޕྋᏊৡ ౦ࢂցቹៜלচϐڰۓૈΚǴஒלচϩձྋܭόӕྋᏊύǴ่݀ᡉҢǴ྽לচྋܭ ᕗለ጗ፂన (phosphate buffer) ܈ HPSEC ࢬࢱన (0.3 M NaNO3 ֖ 0.02% NaN3)Ǵ ځ֎Ӏॶᆶᐚࡋևጕ܄ᜢ߯ǴऩஒלচྋܭΒԛᇃᚖНǴ߾όڀԖጕ܄ᜢ߯Ǵ௢ෳ

ࢂྋᏊύޑᡶᜪᚆηԖշܭלচڰۓܭ 96 Ͼዬ (კΜΐ-კΒΜ΋)Ƕ

! ! לচڰۓܭ༟਑߄य़ϐ֎ߕϸᔈࢂᙖҗஒלচྋܭեᚆηமࡋޑᡵ܄጗ፂྋ న(Voller et al., 1979)ǴלচᙖҗᒃНϷ౧Н࣬ϕբҔǴڗ،ܭ coating buffer ޑ pH ॶᆶᚆηமࡋ(Shirahama and Suzawa, 1985)Ƕջ٬೚ӭੰࢥלচёаҥջ֎ߕԿ༟

਑߄य़(Voller et al., 1979)Ǵ፾྽ޑ coating buffer ىаߦ຾לচ֎ߕԿ༾ϾዬǴԶל চ֎ߕܭ༟਑߄य़ޑำࡋ߾ޔௗቹៜלচ،ۓՏ࿼ (epitope) ޑཞѨᆶց(Bruck et

al., 1982; McCullough et al., 1985)Ƕࣁࡌҥ coating buffer ύ pH ॶᆶᚆηமࡋჹܭ

לচ֎ߕܭ༾ϾዬϐቹៜǴჴᡍаᕗለ጗ፂనࣁЬǴׯᡂ pH ॶᆶ NaCl ᐚࡋ຾Չ

ࣴزǶ

! ! ٬ҔѤᅿ኱ྗࠔϷჹᔈϐלᡏǶGum arabic Ьाࣁ arabinogalactanǴڀԖ proteinǴ ࣁ΋ፄᚇ่ᄬǴᒣᇡޑלᡏࣁ LM2ǶCitrus pectinǴڀԖ 85% GalAǴ֖Ԗεໆለ܄

ᑗǴჹᔈϐלᡏࣁ LM5Ƕ4-O-methyl-glucuronoxylanǴа LM10 לᡏᆶϐᒣᇡǶӧ gum arabic ᆶ LM2 ᒃکΚϸᔈύǴpH ॶᆶלচ֎ߕόڀ҅࣬ᜢ܄ǶCitrus pectin ࣁ

ለ܄ᑗǴᕗለ጗ፂనܭ pH ॶ฻ܭ 6.4 ਔԖനଯޑ֎ӀॶǴЪܴᡉӧ pH ॶࣁ 3.4 Կ 6.4 ϐለ܄ᕉნΠځ֎ӀॶၨமᡵᕉნࣁଯǴ௢ෳҗܭለ܄ᑗޑ pKa=3-5Ǵܭለ܄

ᕉნΠځႝ಻೏፿ጨǴ৒ܰ֎ߕܭ 96 ϾዬǶឦܭύ܄ᑗޑ(1ʈ5)-α-L-arabinan ᆶ xylose: GlcA=10:1 ޑ 4-O-methyl-glucuronoxylanǴҗܭᎉለਥᚆηޑ pKa=3-5Ǵӧ pH 6.4-9.4 ޑᕉნΠᎉለਥှᚆࣁ COO^ˇǴԖճ֎ߕܭ༾ϾዬǶ྽ᕉნύϐ pH ॶ ౦୏ਔǴ཮ׯᡂלচ่ᄬϐႝ಻܄Ϸᆶ༾ϾዬϐѾΚǴ຾Զቹៜלচᆶ༾Ͼዬޑ֎

ߕ (კΒΜΒ)Ƕ

! ! ֎ߕϸᔈёᙖҗ NaCl ᐚࡋ΢ϲԶගϲǴቚуᚆηமࡋჹלচ֎ߕܭӭኧ౧Н ᆫӝނӵ polystyrene Ԗ҅࣬ᜢ܄ǴԶ polystyrene Ψࢂ 96 ϾዬޑЬा׷਑Ƕࣁ௖૸

ᡶࡋჹלচ֎ߕޑቹៜǴ٬Ҕόӕ NaCl ᐚࡋޑᕗለ጗ፂనǶӧ gum arabic ᆶ LM2 ᒃکΚϸᔈύǴNaCl ᐚࡋᆶלচ֎ߕόڀ҅࣬ᜢǶCitrus pectin ޑ֎ߕΚڙ҂బу NaCl ޑಔձቹៜၨځдಔձଯǴځ֎ӀॶܴᡉၨեǴԶځᎩᡶࡋᡂϯჹܭځ֎ߕ բҔคᡉ๱ৡ౦Ƕ0-0.8M ϐᡶࡋճܭ (1ʈ5)-α-L-arabinanǴࠅ෧Ͽ citrus pectin ᆶ 4-O-methyl-glucuronoxylan ฻஥Ԗለ܄ᑗϐ֎ߕǶ3.2M ᆶ 4.0M NaCl ϐଯᡶࡋё

ܴᡉቚу 4-O-methyl-glucuronoxylan ֎ߕǶቚу֎ߕϸᔈޑЬӢ௢ෳࣁלচϷ༾Ͼ ዬ໔ޑᓉႝΚ (electrostatic) ᆶѾΚ (repulsive force) ӢᚆηமࡋቚуԶύکǴቚ уלচ֎ߕ (კΒΜΟ)Ƕ

კΜΐǵόӕᐚࡋ gum arabic ྋܭΒԛᇃᚖНᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ݋

(R²=0.1586)Ƕ

Figure 19. Immunoaffinity of LM2 on gum arabic in ddH2O with different concentrations (R²=0.1586).

კΒΜǵόӕᐚࡋ gum arabic ྋܭᕗለ጗ፂనᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ݋

(R²=0.9924)Ƕ

Figure 20. Immunoaffinity of LM2 on gum arabic in phosphate buffer with different concentrations (R²=0.9924).

კΒΜ΋ǵόӕᐚࡋ gum arabic ྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02%

NaN3) ᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ݋ (R²=0.998)Ƕ

Figure 21. Immunoaffinity of LM2 on gum arabic in HPSEC eluent (0.3M NaNO3and 0.02% NaN3) with different concentrations (R²=0.998).

1Values followed by different letters in the same group are significantly different (Duncan’s test p<0.05).

კΒΜΒǵόӕ pH ॶϐᕗለ጗ፂనჹܭ gum arabicǵcitrus pecitnǵ(1ʈ5)-α-L-arabinan ᆶ 4-O-methyl-glucuronoxylan Ϸൂਲ਼לᡏ LM2ǵLM5ǵLM6 ᆶ LM10 ϐ

֎ӀॶቹៜǶ

Figure 22. Immunoaffinity of LM2, LM5, LM6 and LM10 on gum arabic, citrus pectin, (1ʈ5)-α-L-arabinan and 4-O-methyl-glucuronoxylan in phosphate buffer with different pH values.

1Values followed by different letters in the same group are significantly different (Duncan’s test p<0.05).

კΒΜΟǵόӕෛϯ໊ᐚࡋϐᕗለ጗ፂనჹܭ gum arabicǵcitrus pecitnǵ(1ʈ5)-α-L-arabinan ᆶ 4-O-methyl-glucuronoxylan Ϸჹᔈϐൂਲ਼לᡏ LM2ǵLM5ǵLM6 ᆶ LM10 ϐ֎ӀॶቹៜǶ

Figure 23. Immunoaffinity of LM2, LM5, LM6 and LM10 on gum arabic, citrus pectin, (1ʈ5)-α-L-arabinan and 4-O-methyl-glucuronoxylan in phosphate buffer with different concentrations of NaCl.

ಃ

ಃΒ࿯ġǵଯᏊໆ㸃ރਏᔈ (High dose hook effect)

! ! ! ! ࣁளޕൂਲ਼לᡏᆶόӕלচᐚࡋ຾ՉխࣝᒃکΚϸᔈϐ֎Ӏॶޑጕ܄ጄ ൎǴஒϖᅿൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵLM10 ᆶ LM20 ᆶჹᔈϐלচ຾Չೱុ

ีញǴ٠ஒלচϩձྋှܭᕗለ጗ፂనϷ HPSEC ࢬࢱన (კΒΜѤ-კΒΜϖ)Ƕ

่݀ᡉҢǴᐚࡋଯܭ 20 μg/mLǴ֎Ӏॶό཮ևጕ܄ᜢ߯Ƕҗ଑ᘜϩ݋ύޑ،ۓ߯

ኧ R²ॶ૸ፕځጕ܄ᜢ߯Ƕ྽ᐚࡋϟܭ 0.1~100 μg/mL ጄൎϣǴR²ॶပܭ 0.08-0.60Ǵ֎Ӏॶᆶלচᐚࡋόڀጕ܄ᜢ߯Ǵ྽לᡏᐚࡋϟܭ 0.1~20 μg/mL ໔ǴR²ॶ

ࣣܭ 0.93 а΢ǴԖၨӳޑጕ܄࣬ᜢ (კΒΜϤ-კΟΜϖ)Ƕଯᐚࡋኬࠔޑ֎Ӏॶ

ၨեᐚࡋኬࠔٰளեǴԜ౜ຝࣁଯᏊໆ㸃ރਏᔈ (high dose hook effect )ǴЬाว ғܭሇનೱ่խࣝ֎ߕϸᔈǴ२ԛว౜ܭ 1974 ԃǴMiles ฻ΓаΓᜪՈమ៓ೈқ

(human serum ferritin) ᆶีញ 500ǵ1000ǵ10000 Ϸ 25000 ७ϐלচ ferritin ຾Չ two site immunoradiometric assayǴ่݀ᡉҢଯᐚࡋלচǴջีញ 500 ७ϐಔձǴ ځ᠐ॶၨځдಔձեǶԜਏᔈҭܭ۳ࡕว౜ܭᖏ׉խࣝᔠෳ಻ᅟᆾᆶဍዦ኱૶Ǵ ӵ macroprolactinoma ύϐ prolactin (St-jean et al., 1996; Frieze et al., 2002), prostate specific antigen(Charrie et al., 1995; Furuya et al., 2001), hepatoblastoma ύ alpha-fetoprotein(Jassam et al., 2006), Ϸ metastatic medullary thyroid carcinoma ύޑ calcitonin (Leboeuf et al., 2006)Ǵ΋ѿלচᐚࡋຬၸלচ،ۓՏ࿼֖ໆǴջ㸃ރ⸣

ॶ (hook threshold)Ǵෞᚆלচ (free antigen) ջ཮کᆶלᡏೱ่ޑלচᝡݾǴࡺ

྽לচᐚࡋቚуਔǴр౜՟ࢂԶߚ (paradoxical) ޑեϸᔈ่݀(Jassam et al., 2006)Ƕவόӕᐚࡋჹᔈ֎Ӏॶϐ่݀ᡉҢǴଯᏊໆ㸃ރਏᔈჹܭ֎Ӏॶϐቹៜཱུ

εǴऩ٬ҔᒃکΚϸᔈ຾ՉۓໆਔǴ໪ஒלচᐚࡋีញܭጕ܄ጄൎϣǶ

კΒΜѤǵόӕᐚࡋ኱ྗࠔྋܭᕗለ጗ፂనᆶൂਲ਼לᡏᒃکΚ่݀Ƕ

Figure 24. Immunoaffinity of monoclonal antibodies on antigens in phosphate buffer with different concentrations. LM2 and gum arabic (Ʌ), LM5 and citrus pectin (Ʉ), LM6 and (1→5)-α-L-arabinan (ɐ*- LM10 and 4-O-methyl-glucuronoxylan (ɏ*-LM20 and citrus pectin (Ɏ*/!

კΒΜϖǵόӕᐚࡋ኱ྗࠔྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02% NaN3) ᆶ

ൂਲ਼לᡏᒃکΚ่݀Ƕ

Figure 25. Immunoaffinity of monoclonal antibodies on antigens in HPSEC eluent (0.3M NaNO3and 0.02% NaN3) with different concentrations. LM2 and gum arabic (Ʌ), LM5 and citrus pectin (Ʉ), LM6 and (1→5)-α-L-arabinan (ɐ*- LM10 and 4-O-methyl-glucuronoxylan (ɏ*- LM20 and citrus pectin (Ɏ*/!

კΒΜϤǵόӕᐚࡋ gum arabic ྋܭᕗለ጗ፂనᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ݋

(R²=0.9993)Ƕ

Figure 26. Immunoaffinity of LM2 on gum arabic in phosphate buffer with different

კΒΜΎǵόӕᐚࡋ gum arabic ྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02%

NaN3) ᆶൂਲ਼לᡏ LM2 ϐᒃکΚϩ݋ (R²=0.9502)Ƕ

Figure 27. Immunoaffinity of LM2 on gum arabic in HPSEC eluent (0.3M NaNO3and 0.02% NaN3) with different concentrations (R²=0.9502).

კΒΜΖǵόӕᐚࡋ citrus pectin ྋܭᕗለ጗ፂనᆶൂਲ਼לᡏ LM5 ϐᒃکΚϩ݋

(R²=0.9993)Ƕ

Figure 28. Immunoaffinity of LM5 and on citrus pectin in phosphate buffer with different concentrations (R²=0.9993).

კΒΜΐǵόӕᐚࡋ citrus pectin ྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02%

NaN3) ᆶൂਲ਼לᡏ LM5 ϐᒃکΚϩ݋ (R²=0.9905)Ƕ

Figure 29. Immunoaffinity of LM5 on citrus pectin in HPSEC eluent (0.3M NaNO3and 0.02% NaN3) with different concentrations (R²=0.9905).

კΟΜǵόӕᐚࡋ(1→5)-α-L-arabinan ྋܭᕗለ጗ፂనᆶൂਲ਼לᡏ LM6 ϐᒃکΚ ϩ݋ (R²=0.9565)Ƕ

Figure 30. Immunoaffinity of LM6 on (1→5)-α-L-arabinan in phosphate buffer with different concentrations (R²=0.9565).

კΟΜ΋ǵόӕᐚࡋ(1→5)-α-L-arabinan ྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02% NaN3) ᆶൂਲ਼לᡏ LM6 ϐᒃکΚϩ݋ (R²=0.9388)Ƕ

Figure 31. Immunoaffinity of LM6 on (1→5)-α-L-arabinan in HPSEC ࢬࢱన (0.3M NaNO3and 0.02% NaN3) with different concentrations (R²=0.9388).

კΟΜΒǵόӕᐚࡋ 4-O-methyl-glucuronoxylan ྋܭᕗለ጗ፂనᆶൂਲ਼לᡏ LM10 ϐᒃکΚϩ݋ (R²=0.9587)Ƕ

Figure 32. Immunoaffinity of LM10 on 4-O-methyl-glucuronoxylan in phosphate buffer with different concentrations (R²=0.9587).

კΟΜΟǵόӕᐚࡋ 4-O-methyl-glucuronoxylan ྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02% NaN3) ᆶൂਲ਼לᡏ LM10 ϐᒃکΚϩ݋ (R²=0.9957)Ƕ

Figure 33. Immunoaffinity of LM10 on 4-O-methyl-glucuronoxylan in HPSEC eluent (0.3M NaNO3and 0.02% NaN3) with different concentrations (R²=0.9957).

კΟΜѤǵόӕᐚࡋ citrus pectin ྋܭᕗለ጗ፂనᆶൂਲ਼לᡏ LM20 ϐᒃکΚϩ݋

(R²=0.996)Ƕ

Figure 34. Immunoaffinity of LM20 on citrus pectin in phosphate buffer with different

კΟΜϖǵόӕᐚࡋ citrus pectin ྋܭ HPSEC ࢬࢱన (0.3M NaNO3֖ 0.02%

NaN3) ᆶൂਲ਼לᡏ LM20 ϐᒃکΚϩ݋ (R²=0.9995)Ƕ

Figure 35. Immunoaffinity of LM20 on citrus pectin in HPSEC eluent (0.3M NaNO3

and 0.02% NaN3) with different concentrations (R²=0.9995).

ಃ

ಃΟകǵġНྋ܄όё੃ϯӭᗐύӚӭᗐ୔ϩϐൂਲ਼לᡏᒃکΚ่݀

! ! Ҟ߻ςԖӭኧޑൂਲ਼לᡏҔаᒣᇡ෌ނಒझᏛ΢όӕޑ݀ጤ่ᄬǴҭว߄݀

ጤ΢ӭᅿלচ،ۓՏ࿼(Willats et al., 2001)ǶҁፕЎ٬Ҕᒣᇡ݀ጤӭᗐϐൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ຾ՉխࣝᒃکϸᔈǶ

! ! F1Ǵᆶ LM5 ڀԖᒃکϸᔈǴൂᗐಔԋЬाࣁ arabinose (18.92%) Ϸ galactose (68.53%)Ǵ௢ෳࣁ arabinogalactan type I ่ᄬ (კΟΜϤ)ǶF2 ୔ϩǴarabinose ᆶ galactose К ٯ ࣁ 1.16:1 Ǵ Ъ ჹ ܭ LM5 ᆶ LM6 ڀ Ԗ ᒃ ک ϸ ᔈ Ǵ ௢ ෳ ࣁ rhamnogalacuronan I (RGI) ΢ϐ arabinogalactan type I (AGI) Ϸ linear arabinan ϩЍ (კΟΜΎ)ǴԜ่݀ёаჹᔈډൂᗐಔԋǶՋࢩୖਥӧ຾Չ௦ԏࡕ཮а໚Ӏᚼᠴ຾

ՉߥӸǴ௢ෳ෌ނύ rhamnogalacturonase ᆶ galacturonase Нှ RG-I ϐЬ༸ᆶϩЍ

೽ϩǴ٬ RGI ϷϩЍ೽ϩᘐ຋ࣁλТࢤǴಔԋ F1 ᆶ F2Ǵό஥ᆶ༾஥ႝ಻ϐ୔ϩǶ

! ! F3 ୔ϩǴҗᒃکϸᔈ่݀௢ෳǴϩηໆεޑ୔ୱࣁ AGI Ϸ AGII ่ᄬǴԶ linear

arabinan ߾ቶݱϩѲǴԜ௢ෳёҗൂᗐಔԋКٯளډᡍ᛾ǶF3 ֖Ԗ 3.55% GlcAǴ ᡉҢࣁ AGII ่ᄬǶarabinose : galactose=1.80:1Ǵऩ໻Ԗ arabinogalactan type I Ϸ type II ่ᄬǴarabinose ֖ໆᔈ၀ό཮ଯܭ galactose ߈ 2 ७ǴځᎩޑ arabinose ёૈ

ࢂҗ linear arabinan ϩЍ܌ගٮ (კΟΜΖ)ǶF4 ୔ϩЬाࣁለ܄ᑗǴჹܭ LM2ǵ LM5ǵLM6 ӕኬڀԖᒃکϸᔈǴΨёவൂᗐКٯளޕځڀԖ AGIǵAGII Ϸ linear arabinan ϐϩЍ่ᄬǴՠࢂ֖ໆܴᡉК F3 եǶF4 Ьाჹܭ LM19 Ԗமਗ਼ϐᒃکϸ ᔈǴF3 ჹځϐૻဦ߾ၨ১ (კΟΜΐ)ǶLM19 ᆶ LM7ǵLM20ǵJIM7 ࣣࣁ anti-homogalacuronan (HG) לᡏǴHG ΢Ҙ୷✊ϯϐำࡋϷՏ࿼߾،ۓלᡏ໔ᒣᇡלচ

،ۓՏ࿼ϐৡ౦ǶClausen ӧ 2003 ԃஒ hexagalacturonan ܭόӕՏ࿼ௗ΢Ҙ୷ (Clausen et al., 2003)ǴLM7 ᒣᇡޑ่ᄬࣁٿঁҘ୷✊ϯ୷ϐ໔ǴѤঁೱុόڀҘ୷

✊ϯޑ galacturonanǶJIM5 (לচ،ۓՏ࿼ӕ LM19)ǴᒣᇡΟঁೱុόڀҘ୷✊ϯ ޑ galacturonanǴԶ JIM7 (לচ،ۓՏ࿼ӕ LM20) ߾ሡԖೱុ܈໔႖௨ӈϐΟԿ Ѥঁ methyl esterified galacturonan ωૈᆶϐᒣᇡǶᕴکٰᇥǴѤᅿ homogalacturonan ޑלᡏᒣᇡޑ partially methyl esterified homogalacturonan Ӛό࣬ӕǴ٩ྣ܌ሡϐ HG Ҙ୷✊ϯำࡋᆶஏ໣ࡋ௨ӈǴҗեԿଯࣁ LM7ǵLM19ǵJIM7ǵLM20Ƕ

ġ ġ F3 ᆶ F4 ୔ϩᆶѤᅿ anti-homogalacturonan לᡏϐᒃک่݀Ǵ໻ჹܭ LM19 ڀ ԖᒃکϸᔈǴ߄Ңٿঁ୔ϩࣣԖ homogalacturonan ่ᄬǴ٠ЪΨࢂൂᗐಔԋύ galacturonic acid ϐЬाٰྍǴҗ LM19 ჹܭלচޑᒣᇡёаளޕǴF3 ᆶ F4 ܌֖

Ԗޑ HG ่ᄬࣁϿໆҘ୷✊ϯϐ homogalacturonanǶHG ଆ߃ࢂаଯҘ୷✊ϯޑރ ᄊ೏ӝԋǴՠࢂ෌ނϣޑ pectin methylesterase ཮ჹܭ HG ຾ՉѐҘ୷✊ϯ (de-esterified)ǴόӕҘ୷✊ϯำࡋ཮ቹៜځғނཀကǴញрޑԾҗ♐୷ (free carboxyl groups) ᆶ໚ᚆηᗖ่܈ᆶ݀ጤᗐ᜘຾ՉҬᖄǴቹៜԋጤ܄፦ᆶ݀ጤӭᗐޑ่ᄬǶ ќ΋Бय़Ǵа KDO ෳۓ F3ǵF4ǴڀԖ II ่ᄬǴԜ่ᄬࢂҗ࿝ለ✊ᆶځд RG-II ่ᄬᖄ่Ǵ׎ԋ RG-RG-II ΒᆫᡏǴࣁಒझᏛமࡋٰྍǴቹៜ݀ጤᖄᆛϐϾࢰЁκϷ ቸ܄Ǵ F1-F4 Ѥঁ୔ϩჹܭ LM10 ࣣόڀԖᒃکϸᔈǴЪൂᗐಔԋύ҂Ԗ xyloseǴ

ࡺԜѤ୔ϩόڀԖ xylan ܈ xylogalacturonan ่ᄬǴԶ xylose ӧ෌ނύЬाӸӧъ ᠼᆢનǴҗԜ௢ፕǴՋࢩୖਥό֖ъᠼᆢનǴځӭᗐЬाࣁ݀ጤӭᗐǶ

! ! а΢่݀ᡉҢǴՋࢩୖНྋ܄όё੃ϯӭᗐޑЬाԋϩࣁ݀ጤӭᗐǴऊԖ 39%

ޑό஥܈༾஥ႝ಻ϐӭᗐёૈࣁ݀ጤӭᗐύڀԖଯࡋϩЍޑ RGI ୔༧ϐफ़ှТࢤǴ ٠௢ෳՋࢩୖӭᗐύ 61%ޑ F3 ᆶ F4 ୔ϩǴࣁ෌ނಒझᏛ݀ጤ่ᄬޑЬाಔԋǴ ٠Ъҗ HGǵRGI ᆶ RGII ่ᄬǴಔԋ݀ጤᖄᆛǴ຾Զ،ۓ߃ભಒझᏛޑமࡋǵቸ

܄ᆶфૈ܄Ƕ

კΟΜϤǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F1 ᆶൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ϐᒃکΚϩ݋Ƕ Figure 36. HPSEC elution profile of fraction one of water-soluble nondigestible polysaccharide of P. quinquefolius on DEAE chromatography combined with the ELISA response of the monoclonal antibody LM2, LM5, LM6, LM7, LM10, LM19, LM20 and JIM7 affinity.

კΟΜΎǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F2 ᆶൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ϐᒃکΚϩ݋Ƕ Figure 37. HPSEC elution profile of fraction two of water-soluble nondigestible polysaccharide of P. quinquefolius on DEAE chromatography combined with the ELISA response of the monoclonal antibody LM2, LM5, LM6,LM7, LM10, LM19, LM20 and JIM7 affinity.

კΟΜΖǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F3 ᆶൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ϐᒃکΚϩ݋Ƕ Figure 38. HPSEC elution profile of fraction three of water-soluble nondigestible polysaccharide of P. quinquefolius on DEAE chromatography combined with the ELISA response of the monoclonal antibody LM2, LM5, LM6,LM7, LM10, LM19,

კΟΜΐǵՋࢩୖНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐ F4 ᆶൂਲ਼לᡏ LM2ǵLM5ǵLM6ǵǵLM7ǵLM10ǵLM19ǵLM20 ᆶ JIM7 ϐᒃکΚϩ݋Ƕ Figure 39. HPSEC elution profile of fraction four of water-soluble nondigestible polysaccharide of P. quinquefolius on DEAE chromatography combined with the ELISA response of the monoclonal antibody LM2, LM5, LM6,LM7, LM10, LM19, LM20 and JIM7 affinity.

߄ѤǵНྋ܄όё੃ϯӭᗐа DEAE ቫ݋ϩᚆϐѤঁ୔ϩڀԖϐ݀ጤ่ᄬ΋ំ

߄Ƕ

Table 4. The pectin structure of the four fractions of water-soluble nondigestible polysaccharide of P. quinquefolius L. on DEAE column

Fraction HG^a XGA^b RGI

arabinan AGI AGII RGII

F1 ˇˇ ˇˇ ˇˇ ˁˁ ˇˇ ˇˇ

F2 ˇˇ ˇˇ ˁˁ ˁˁ ˇˇ ˇˇ

F3 ˁˁ ˇˇ ˁˁ ˁˁ ˁˁ ˁˁ

F4 ˁˁ ˇˇ ˁˁ ˁˁ ˁˁ ˁˁ

Defined by the data of monoclonal polysaccharide composition and the immunoaffinity with mAbs and KDO determination.

aHomogalacturonan.

bXylogalacturonan.

ഌ

ഌǵġ่ፕ

! ! ҁࣴز่ӝሇનНှǵ഍ᚆηᐋિቫ݋ǵଯਏૈϩηᑔቫ݋ݤᆶൂਲ਼לᡏխࣝ

ᒃکϸᔈѤᅿמೌǴаՋࢩୖࣁෳ၂চ਑Ǵࡌҥёӕ؁ϩ݋ӭᅿНྋ܄ӭᗐޑמೌ

ѳѠǶ

! ! Ջࢩୖಉӭᗐа 95.7%ϐ 1,4;1,6-α-D-glucan ё੃ϯӭᗐǴᆶ 4.3%Нྋ܄ό ё੃ϯӭᗐ܌ಔԋǶНྋ܄όё੃ϯӭᗐ࿶഍ᚆηҬඤᐋિቫ݋ϩᚆёϩࣁ F1ǵ F2ǵF3 ᆶ F4 Ѥঁ୔ϩǶҁࣴز٬Ҕଯਏૈϩηᑔቫ݋س಍ᆶൂਲ਼לᡏᒃکϸᔈམ ଛൂᗐಔԋ௢ෳНྋ܄όё੃ϯӭᗐѤঁ୔ϩϐ่ᄬǶ

! ! לচޑ֎ߕϸᔈ཮ቹៜ ELISA ่݀ǶGum arabic ྋܭΒԛᇃᚖНύคݤ֎ߕ

在文檔中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 (頁 23-113)