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Hababka cusub ee difaaca jirka iyo tirada badan ee falanqaynta adag ee oligosaccharides-ka joogtada ah ee ku jira shooladda galleyda ee hore loogu daaweeyay AFEX. Lignocellulosic biomass waa beddel waara oo loogu talagalay shidaalka fosil-ka waxaana si weyn loogu isticmaalaa horumarinta bayotechnologies-ka si loo soo saaro alaabada sida cuntada, quudinta, shidaalka iyo kiimikooyinka. Furaha tiknoolajiyadan waa horumarinta hababka kharashka-ku-saleysan ee loogu beddelayo karbohaydraytyada adag ee ku jira derbiyada unugyada dhirta sonkor fudud sida gulukooska, xylose iyo arabinose. Sababtoo ah biomass-ka lignocellulosic waa mid aad u adag, waa in lagu sameeyaa daaweyn heerkulbeeg ah (tusaale ahaan, xoqidda fiber-ka ammonia (AFEX), asiidhyada khafiifka ah (DA), dareereyaasha ionic (IL)) iyo daawaynta bayoolojiga (tusaale ahaan, hydrolysis-ka enzymatic iyo halsano microbial) si isku dhafan si loo helo badeecada la rabo. Si kastaba ha ahaatee, marka ensaymada fangaska ganacsiga loo isticmaalo habka hydrolysis-ka, 75-85% oo keliya sonkorta milmi karta ee la sameeyay waa monosaccharides, 15-25%-na waa oligosaccharides milmi kara oo aan la xakamayn karin, kuwaas oo aan had iyo jeer loo heli karin noolaha yaryar. Horey, waxaan si guul leh u kala soocnay oo u nadiifinay oligosaccharides-ka adag ee milmi kara iyadoo la adeegsanayo isku-darka kala-soocidda kaarboon iyo diatomaceous dhulka iyo chromatography-ga ka-saarista cabbirka, sidoo kalena waxaan baarnay sifooyinkooda xakamaynta ensaymka. Waxaan ogaanay in oligosaccharides-ka ka kooban beddelka uronic acid ee methylated methylated (DP) ay aad u adag tahay in lagu farsameeyo isku-darka ensaymka ganacsiga marka loo eego oligosaccharides-ka DP ee hooseeya iyo oligosaccharides-ka dhexdhexaadka ah. Halkan waxaan ku soo sheegaynaa isticmaalka dhowr hab oo dheeraad ah, oo ay ku jiraan muuqaalka glycan iyadoo la adeegsanayo unugyada difaaca jirka ee monoclonal (mAbs) oo gaar u ah glycans-ka biomass-ka dhirta si loo garto isku-xidhka glycan ee derbiyada unugyada dhirta iyo hydrolysates-ka enzymatic, ionization-ka laser-ka ee matrix-ka caawiya, spectrometry-ka waqtiga-duulimaadka duulimaadka. . MALDI-TOF-MS) wuxuu adeegsadaa meelaha ugu sarreeya ee ogaanshaha macluumaadka qaab-dhismeedka ee laga helo spectroscopy ka dib burburka labaad ee ions-ka taban, chromatography-ga gaaska iyo spectrometry-ka tirada badan (GC-MS) si loo garto isku-xidhka oligosaccharides-ka oo leh iyo aan lahayn. Sababtoo ah cabbirka yar ee oligosaccharides (DP 4–20), molecules-kan way adag tahay in loo isticmaalo isku xidhka iyo qeexidda mAb. Si aan uga gudubno dhibaatadan, waxaan adeegsannay hab cusub oo ku salaysan isku xidhka biotin kaas oo si guul leh u calaamadeeyay inta badan oligosaccharides-ka DP ee milma ee hooseeya dusha sare ee microplate, kaas oo markaa loo isticmaalay nidaam mAb oo awood badan leh si loo falanqeeyo isku xidhka gaarka ah. Habkan cusub wuxuu sahli doonaa horumarinta tijaabooyinka glycome-ka ee awood badan mustaqbalka kuwaas oo loo isticmaali karo in lagu kala saaro oo lagu garto oligosaccharides-ka ku jira biomarkers ujeeddooyin ogaanshaha.
Biomass-ka Lignocellulosic, oo ka kooban beeraha, kaymaha, cawska iyo walxaha alwaaxda, waa quudin suurtagal ah oo loogu talagalay soo saarista alaabada ku salaysan bayoolojiga, oo ay ku jiraan cuntada, quudinta, shidaalka iyo horudhacyada kiimikada si loo soo saaro badeecooyin qiimo sare leh1. Kaarboohaydraytyada (sida cellulose iyo hemicellulose) ee ku jira derbiyada unugyada dhirta waxaa loo kala saaraa monosaccharides iyadoo la adeegsanayo farsamaynta kiimikada iyo isbeddelka bayoolojiga (sida hydrolysis-ka enzymatic iyo halsano microbial). Daaweynta hore ee caadiga ah waxaa ka mid ah ballaarinta fiber-ka ammonia (AFEX), acid-ka khafiifinta (DA), dareeraha ionic (IL), iyo qaraxa uumiga (SE), kuwaas oo adeegsada isku-darka kiimikooyinka iyo kulaylka si loo yareeyo wax soo saarka lignocellulose iyadoo la furayo derbiyada unugyada dhirta3,4. madax adag oo walxaha ah, 5. Hydrolysis-ka enzymatic waxaa lagu sameeyaa culays adag oo sarreeya iyadoo la adeegsanayo ensaymes-ka firfircoon ee ka kooban karbohaydraytyada ganacsiga (CAZymes) iyo halsano microbial iyadoo la isticmaalayo khamiirka transgenic ama bakteeriyada si loo soo saaro shidaal iyo kiimikooyin ku salaysan bayoolojiga 6.
CAZymes-ka ku jira ensaymyada ganacsiga waxay ka kooban yihiin isku-darka isku-dhafan ee ensaymyada kuwaas oo si isku-dhafan u kala jabiya isku-xidhka karbohaydrayt-sonkorta ee adag si ay u sameeyaan monosaccharides2,7. Sida aan hore u soo sheegnay, shabakadda isku-dhafka ah ee polymers-ka udgoonka leh ee lignin oo leh karbohaydraytyada ayaa ka dhigaysa kuwo aad u dhib badan, taas oo horseedda beddel sonkor oo aan dhammaystirnayn, oo ururisa 15-25% oligosaccharides-ka galmada ee aan la soo saarin inta lagu jiro hydrolysis-ka enzymatic ee biomass-ka hore loo daaweeyay. Tani waa dhibaato caadi ah oo la xiriirta habab kala duwan oo daaweynta biomass-ka ka hor ah. Qaar ka mid ah sababaha caqabaddan waxaa ka mid ah joojinta ensaym-ka inta lagu jiro hydrolysis, ama maqnaanshaha ama heerarka hoose ee ensaymyada muhiimka ah ee lagama maarmaanka ah ee loo baahan yahay si loo jebiyo xiriirka sonkorta ee biomass-ka dhirta. Fahmidda halabuurka iyo astaamaha qaab-dhismeedka ee sonkorta, sida isku-xidhka sonkorta ee oligosaccharides, ayaa naga caawin doona inaan horumarinno beddelka sonkorta inta lagu jiro hydrolysis, taasoo ka dhigaysa hababka bayotechnolojiyada mid tartan kula jira alaabada laga soo saaro batroolka.
Go'aaminta qaab-dhismeedka karbohaydraytyadu waa mid adag waxayna u baahan tahay isku-darka habab sida chromatography dareere ah (LC)11,12, spectroscopy-ga resonance magnetic nuclear (NMR)13, electrophoresis capillary (CE)14,15,16 iyo mass spectrometry (MS)17. ,18. Hababka MS sida spectrometry-ka mass-ka waqtiga-duulaya oo leh laysarka desorption iyo ionization iyadoo la adeegsanayo matrix (MALDI-TOF-MS) waa hab badan oo lagu aqoonsan karo qaab-dhismeedka karbohaydraytyada. Dhawaan, MS-ka isku dhaca ee kala-goynta isku dhaca (CID) ee tandem MS ee sodium ion adducts ayaa si weyn loo isticmaalay si loo aqoonsado faraha faraha ee u dhigma boosaska ku lifaaqan oligosaccharide, habaynta anomeric, taxanaha, iyo boosaska laanta 20, 21.
Falanqaynta Glycan waa qalab aad u fiican oo lagu aqoonsado qoto dheer oo ku saabsan isku xirnaanta karbohaydraytyada22. Habkani wuxuu adeegsadaa unugyada difaaca jirka ee monoclonal (mAbs) oo loo jiheeyay glycan-ka derbiga unugyada dhirta sida baaritaanno si loo fahmo isku xirnaanta karbohaydraytyada adag. In ka badan 250 mAbs ayaa laga heli karaa adduunka oo dhan, oo loogu talagalay oligosaccharides-ka toosan iyo kuwa laanta ah iyadoo la adeegsanayo saccharides-ka kala duwan24. Dhowr mAbs ayaa si weyn loo isticmaalay si loo qeexo qaab-dhismeedka, halabuurka, iyo wax-ka-beddelka derbiga unugyada dhirta, maadaama ay jiraan kala duwanaansho muhiim ah oo ku xiran nooca unugyada dhirta, xubinta, da'da, marxaladda horumarka, iyo deegaanka koritaanka25,26. Dhawaanahan, habkan waxaa loo isticmaalay in lagu fahmo dadka vesicle-ka ee ku jira nidaamyada dhirta iyo xayawaanka iyo doorarkooda ku aaddan gaadiidka glycan sida lagu go'aamiyay calaamadaha unugyada hoose, marxaladaha horumarka, ama kicinta deegaanka, iyo in la go'aamiyo dhaqdhaqaaqa ensaymikada. Qaar ka mid ah qaab-dhismeedka kala duwan ee glycans iyo xylans ee la aqoonsaday waxaa ka mid ah pectin (P), xylan (X), mannan (M), xyloglucans (XylG), glucans isku dhafan (MLG), arabinoxylan (ArbX), galactomannan (GalG), glucuronic acid-arabinoxylan (GArbX) iyo arabino-galactan (ArbG)29.
Si kastaba ha ahaatee, inkastoo dhammaan dadaalladan cilmi-baarista ah, dhowr daraasadood oo keliya ayaa diiradda saaray dabeecadda ururinta oligosaccharide inta lagu jiro hydrolysis-ka culeyska adag ee sare (HSL), oo ay ku jiraan sii deynta oligosaccharide, isbeddellada dhererka silsiladda oligomeric inta lagu jiro hydrolysis, polymers-ka DP ee hooseeya, iyo qaloocyadooda. qaybinta 30,31,32. Dhanka kale, inkastoo falanqaynta glycan ay caddeysay inay tahay qalab waxtar leh oo loogu talagalay falanqaynta dhammaystiran ee qaab-dhismeedka glycan, way adag tahay in la qiimeeyo oligosaccharides-ka DP ee hooseeya ee biyaha ku milma iyadoo la adeegsanayo hababka antibody-ga. Oligosaccharides-ka DP ee yar yar oo leh miisaan molecular ah oo ka yar 5-10 kDa kuma xidhmaan taarikada ELISA 33, 34 waana la dhaqay ka hor inta aan lagu darin antibody-ga.
Halkan, markii ugu horreysay, waxaan ku soo bandhigaynaa tijaabo ELISA ah oo ku saabsan saxamada dahaarka leh ee avidin iyadoo la adeegsanayo unugyada difaaca jirka ee monoclonal, iyadoo la isku darayo habka biotinylation-ka hal-tallaabo ah ee oligosaccharides-ka aan milmi karin iyo falanqaynta glycome-ka. Habkayaga falanqaynta glycome waxaa lagu xaqiijiyay falanqaynta ku salaysan MALDI-TOF-MS iyo GC-MS ee isku xirka oligosaccharide ee dhammaystirka ah iyadoo la adeegsanayo kala soocidda trimethylsilyl (TMS) ee isku-darka sonkorta ee hydrolyzed. Habkan cusub waxaa loo samayn karaa hab wax-soo-saar sare mustaqbalka waxaana laga heli karaa codsi ballaaran oo cilmi-baaris bayooloji ah35.
Wax ka beddelka tarjumaadda kadib ee enzymes-ka iyo unugyada difaaca jirka, sida glycosylation,36 waxay saameeyaan dhaqdhaqaaqooda bayoolojiga. Tusaale ahaan, isbeddellada ku yimaada glycosylation-ka borotiinada serum-ka ayaa door muhiim ah ka ciyaara xanuunka kala-goysyada, isbeddellada ku yimaada glycosylation-ka waxaa loo isticmaalaa calaamadaha ogaanshaha37. glycans kala duwan ayaa lagu soo warramey suugaanta inay si fudud ugu muuqdaan cudurro kala duwan, oo ay ku jiraan cudurrada daba-dheeraada ee bararka caloosha iyo mindhicirka iyo beerka, caabuqyada fayraska, kansarka ugxan-sidaha, naasaha, iyo qanjirka 'prostate'38,39,40. Fahmidda qaab-dhismeedka glycans iyadoo la adeegsanayo hababka glycan ee ku salaysan antibody-ga ELISA waxay siin doontaa kalsooni dheeraad ah oo ku saabsan ogaanshaha cudurka iyada oo aan la isticmaalin hababka adag ee MS.
Daraasaddeennii hore waxay muujisay in oligosaccharides-ka madax adag aan la fuuq-bixin ka dib markii la daaweeyay kahor iyo hydrolysis enzymatic ah (Jaantuska 1). Shaqadayadii hore loo daabacay, waxaan soo saarnay hab soo saarista dhuxusha adag ee firfircoon si loo kala saaro oligosaccharides-ka laga soocay AFEX-ka lagu daweeyay galleyda hydrolyzate (ACSH)8. Ka dib soo saarista iyo kala-soocidda bilowga ah, oligosaccharides-ka waxaa si dheeraad ah loogu kala qaybiyay chromatography-ga ka-saarista cabbirka (SEC) waxaana loo ururiyay siday u kala horreeyaan miisaanka molecular-ka. Monomers-ka sonkorta iyo oligomers-ka laga soo saaray daaweyn kala duwan ayaa lagu falanqeeyay falanqaynta halabuurka sonkorta. Marka la barbar dhigo waxa ku jira oligomers-ka sonkorta ee laga helay habab kala duwan oo daaweyn hore ah, joogitaanka oligosaccharides-ka madax adag waa dhibaato caadi ah oo ku jirta beddelka biomass-ka una beddelo monosaccharides waxayna horseedi kartaa hoos u dhac ku yimaada wax soo saarka sonkorta ugu yaraan 10-15% iyo xitaa ilaa 18%. Mareykanka. Habkan waxaa loo isticmaalaa wax soo saar ballaaran oo dheeraad ah oo jajabyada oligosaccharides ah. ACH-ka ka dhashay iyo jajabyada ku xiga ee leh miisaanno molecular oo kala duwan ayaa loo isticmaalay sidii walxo tijaabo ah oo loogu talagalay qeexidda oligosaccharides-ka shaqadan.
Ka dib markii la daaweeyay kahor iyo laydhka enzymatic-ka, oligosaccharides-ka joogtada ah ayaa aan la hawo-siin. Halkan (A) waa hab kala soocid oligosaccharides ah oo oligosaccharides laga soocay AFEX-ka hore loo daweeyay ee shooladda galleyda hydrolysate (ACSH) iyadoo la isticmaalayo sariir baakaysan oo kaarboon firfircoon iyo dhul diatomaceous ah; (B) Habka kala soocidda oligosaccharides. Oligosaccharides-ka waxaa si dheeraad ah loogu kala saaray chromatography-ga ka saarista cabbirka (SEC); (C) Monomers-ka Saccharide iyo oligomers-ka laga sii daayay daaweyn kala duwan (acid la qasay: DA, dareere ionic ah: IL iyo AFEX). Xaaladaha hydrolysis-ka enzymatic-ka: rarida adag oo sare oo ah 25% (w/w) (qiyaastii 8% rarida glucan), 96 saacadood oo hydrolysis ah, rarida ensaymka ganacsiga 20 mg/g (Ctec2:Htec2:MP-2:1:1 saamiga) iyo (D) Monomers-ka sonkorta iyo oligomers-ka gulukooska, xylose iyo arabinose oo laga sii daayay shooladda galleyda ee hore loo daweeyay ee AFEX (ACS).
Falanqaynta Glycan waxay caddeysay inay tahay qalab waxtar leh oo loogu talagalay falanqaynta qaab-dhismeedka dhammaystiran ee glycans-ka ee laga soo saaray haraaga biomass-ka adag. Si kastaba ha ahaatee, saccharides-ka biyaha ku milma si yar ayaa loo matalaa iyadoo la adeegsanayo habkan dhaqameed41 sababtoo ah oligosaccharides-ka miisaanka molecular-ka hooseeya way adag tahay in lagu dhaqaajiyo saxannada ELISA waxaana la dhaqaa ka hor inta aan lagu darin antibody-ga. Sidaa darteed, isku xidhka antibody-ga iyo sifeynta, habka biotinylation-ka hal-tallaabo ah ayaa loo isticmaalay in lagu dahaadho oligosaccharides-ka milma, ee aan waafaqsanayn saxannada ELISA ee avidin-ku dahaadhay. Habkan waxaa lagu tijaabiyay iyadoo la adeegsanayo ACSH-geenna hore loo soo saaray iyo jajab ku salaysan miisaankiisa molecular (ama heerka polymerization, DP). Biotinylation-ka hal-tallaabo ah ayaa loo isticmaalay in lagu kordhiyo isku xidhka oligosaccharide iyadoo lagu darayo biotin-LC-hydrazide dhammaadka yareynaya ee karbohaydraytka (Jaantuska 2). Xalka, kooxda hemiacetal ee dhammaadka yaraynaya waxay la falgashaa kooxda hydrazide ee biotin-LC-hydrazide si ay u sameeyaan isku xidhka hydrazone. Iyadoo uu joogo wakiilka yareeya NaCNBH3, isku-xidhka hydrazone wuxuu hoos ugu dhacaa badeecad dhammaystiran oo biotinylated ah oo deggan. Iyadoo wax laga beddelayo dhammaadka yareeya sonkorta, ku xidhitaanka oligosaccharides-ka DP ee hooseeya ee saxannada ELISA ayaa suurtogal noqotay, daraasaddayadana tan waxaa lagu sameeyay saxannada dahaarka leh ee avidin iyadoo la adeegsanayo mAbs-ka bartilmaameedsan ee glycan.
Baaritaanka unugyada difaaca jirka ee monoclonal oo ku salaysan ELISA si loogu sameeyo oligosaccharides-ka biotinylated. Halkan (A) isku-darka biotinylation ee oligosaccharides iyo baaritaanka ELISA ee xiga oo leh mAbs-ka bartilmaameedsan ee glycan ee saxannada dahaarka leh ee NeutrAvidin iyo (B) waxay muujinaysaa hab hal-tallaabo ah oo loogu talagalay biotinylation-ka alaabada falcelinta.
Saxannada dahaarka leh ee Avidin oo leh unugyada difaaca jirka ee isku xiran ee oligosaccharide ayaa markaa lagu daray unugyada difaaca jirka ee aasaasiga ah iyo kuwa sare waxaana lagu dhaqay meel iftiin iyo waqti-xasaasi ah. Ka dib marka la dhammeeyo isku xidhka unugyada difaaca jirka, ku dar substrate-ka TMB si aad u kululeyso saxanka. Falcelinta waxaa ugu dambeyntii lagu joojiyay aashitada sulfuric. Saxannada la dahaadhay waxaa lagu falanqeeyay akhristaha ELISA si loo go'aamiyo xoogga isku xidhka ee unugyada difaaca jirka kasta si loo ogaado isku xidhka gaarka ah ee unugyada difaaca jirka. Wixii faahfaahin ah iyo xuduudaha tijaabada, fiiri qaybta u dhiganta "Qalabka iyo Hababka".
Waxaan muujineynaa faa'iidada habkan cusub ee loo sameeyay codsiyada gaarka ah annagoo sifeynayna oligosaccharides-ka milma ee ku jira ACSH iyo sidoo kale jajabyada oligosaccharides-ka cayriin iyo kuwa la safeeyey ee laga soocay lignocellulosic hydrolysates. Sida ku cad Jaantuska 3, xylans-ka ugu badan ee epitope-ka lagu beddelo ee lagu garto ACSH iyadoo la adeegsanayo hababka baaritaanka glycome-ka bioacylated waa badanaa uronic (U) ama methyluronic (MeU) iyo pectic arabinogalactans. Inta badan waxaa sidoo kale laga helay daraasaddeenna hore ee ku saabsan falanqaynta glycans-ka ee adag ee aan hydrolyzed (UHS)43.
Ogaanshaha epitopes-ka oligosaccharide ee dib u soo celiya iyadoo la adeegsanayo antibody monoclonal ah oo loo jiheeyay glycan-ka derbiga unugga. Jajabka "dhexdhexaadka ah" waa jajabka ACN, jajabka "asiidhka" waa jajabka FA. Casaanka dhalaalaya ee khariidadda kulaylka ayaa tilmaamaya wax badan oo epitope ah, buluugga dhalaalayana wuxuu muujinayaa asal madhan. Qiimaha midabka ee miisaanka ku jira waxay ku salaysan yihiin qiimayaasha OD cayriin ee qaacidooyinka N=2. Epitopes-ka ugu muhiimsan ee ay aqoonsadeen antibody-yadu waxaa lagu muujiyay midigta.
Qaab-dhismeedkan aan ahayn cellulose-ka laguma kala jari karo cellulases-ka ugu badan iyo hemicellulases-ka isku darka ensaymka ganacsiga ee la tijaabiyay, kaas oo ay ku jiraan ensaymada ganacsiga ee ugu badan ee la isticmaalo. Sidaa darteed, ensaymada cusub ee kaabayaasha ah ayaa looga baahan yahay hydrolysis-kooda. Iyadoo aan lahayn ensaymada dheeraadka ah ee aan ahayn cellulose-ka ee lagama maarmaanka ah, isku-xidhyadan aan ahayn cellulose-ka waxay ka hortagayaan in si buuxda loogu beddelo monosaccharides, xitaa haddii polymer-kooda sonkorta ee waalidkood si ballaaran loogu shubo jajabyo gaagaaban oo lagu milmo iyadoo la isticmaalayo isku-darka ensaymka ganacsiga.
Daraasad dheeraad ah oo ku saabsan qaybinta calaamadaha iyo xooggeeda isku xidhka ayaa muujisay in epitopes-ka isku xidhka ay ka hooseeyaan jajabyada sonkorta DP ee sare (A, B, C, DP ilaa 20+) marka loo eego jajabyada DP ee hooseeya (D, E, F, DP) ee dimers) (Jaantuska 1). Jajabyada aashitada ayaa aad ugu badan epitopes-ka aan cellulose ahayn marka loo eego jajabyada dhexdhexaadka ah. Ifafaalahan waxay la jaan qaadayaan qaabka lagu arkay daraasaddeenna hore, halkaas oo qaybaha sare ee DP iyo aashitada ay aad uga adkaysi badnaayeen hydrolysis-ka enzymatic. Sidaa darteed, joogitaanka epitopes-ka aan cellulose ahayn ee glycan iyo beddelka U iyo MeU waxay si weyn uga qayb qaadan karaan xasilloonida oligosaccharides. Waa in la ogaadaa in hufnaanta isku xidhka iyo ogaanshaha ay dhibaato ku noqon karto oligosaccharides-ka DP ee hooseeya, gaar ahaan haddii epitope-ku yahay dimeric ama trimeric oligosaccharides. Tan waxaa lagu tijaabin karaa iyadoo la isticmaalayo oligosaccharides-ka ganacsiga ee dherer kala duwan, mid walbana wuxuu ka kooban yahay hal epitope oo ku xidhan mAb gaar ah.
Sidaa darteed, isticmaalka unugyada difaaca jirka ee qaab-dhismeedka gaarka ah ayaa muujiyay noocyo gaar ah oo ah isku xidhka dib-u-celinta. Iyada oo ku xidhan nooca unugyada difaaca jirka ee la isticmaalay, qaabka isku xidhka ee ku habboon, iyo xoogga calaamadda ay soo saarto (ugu badnaan iyo ugu yar), enzymes-ka cusub ayaa la aqoonsan karaa oo lagu dari karaa nus-tiro ahaan isku darka enzyme-ka si loo helo glycoconversion dhammaystiran. Marka la eego falanqaynta oligosaccharides-ka ACSH tusaale ahaan, waxaan abuuri karnaa xog ururin ah oo ku saabsan isku xidhka glycan ee walxo kasta oo biomass ah. Waa in halkan lagu xuso in isku xidhka kala duwan ee unugyada difaaca jirka la tixgeliyo, haddii aan la garanayn isku xidhkooda, tani waxay abuuri doontaa dhibaatooyin gaar ah marka la barbar dhigayo calaamadaha unugyada difaaca jirka ee kala duwan. Intaa waxaa dheer, isbarbardhigga isku xidhka glycan ayaa laga yaabaa inuu si fiican ugu shaqeeyo muunadaha isku xidhka antibody-ga. Xidhiidhadan adag ayaa markaa lagu xidhi karaa xogta CAZyme, kaas oo aan ka aqoonsan karno enzymes-ka, aan ka xulan karno enzymes-ka musharaxa ah oo aan ku tijaabin karno enzymes-ka jebinta isku xidhka, ama aan horumarinno nidaamyada microbial-ka si loo muujiyo ensaymadan si loogu isticmaalo biorefineries44.
Si loo qiimeeyo sida hababka difaaca jirka ay u dhammaystiraan hababka kale ee lagu garto oligosaccharides miisaanka yar ee molecular-ka ee ku jira lignocellulosic hydrolysates, waxaan sameynay MALDI (Jaantuska 4, S1-S8) iyo falanqaynta saccharides-ka laga soo qaatay TMS iyadoo lagu saleynayo GC-MS isla guddiga (Jaantuska 5) ee oligosaccharide. MALDI waxaa loo isticmaalaa in lagu barbardhigo in qaybinta tirada ee molecules-ka oligosaccharide ay la mid tahay qaab-dhismeedka loogu talagalay. Sawirka 4 wuxuu muujinayaa MS ee qaybaha dhexdhexaadka ah ee ACN-A iyo ACN-B. Falanqaynta ACN-A waxay xaqiijisay noocyo kala duwan oo sonkor ah oo pentose ah oo u dhexeeya DP 4-8 (Jaantuska 4) ilaa DP 22 (Jaantuska S1), oo miisaankoodu u dhigmayo oligosaccharides-ka MeU-xylan. Falanqaynta ACN-B waxay xaqiijisay taxanaha pentose iyo glucoxylan ee DP 8-15. Qalabka dheeraadka ah sida Jaantuska S3, khariidadaha qaybinta cufnaanta aashitada ee FA-C waxay muujinayaan noocyo kala duwan oo sonkor ah oo pentose ah oo lagu beddelay (Me)U oo leh DP ah 8-15 oo la jaan qaadaya xylan-yada la beddelay ee laga helo baaritaanka mAb ee ku salaysan ELISA. Epitopes-ku waa isku mid.
Kala duwanaanshaha MALDI-MS ee oligosaccharides-ka milmi kara ee aan waafaqsanayn ee ku jira ACS. Halkan, (A) Qaybaha miisaanka hooseeya ee ACN-A oo ka kooban methylated uronic acid (DP 4-8) oo lagu beddelay glucuroxylan oligosaccharides iyo (B) ACN-B xylan iyo methylated uronic acid oligosaccharides oo lagu beddelay glucuroxylan (DP 8-15).
Falanqaynta halabuurka haraaga glycan ee oligosaccharides-ka aan la qabsan karin. Halkan (A) Halabuurka TMS saccharides ee jajabyada oligosaccharide ee kala duwan ee laga helay falanqaynta GC-MS. (B) Qaab-dhismeedka sonkorta kala duwan ee laga soo qaatay TMS ee ku jirta oligosaccharides. ACN - jajabka acetonitrile oo ka kooban oligosaccharides dhexdhexaad ah iyo jajabka FA - ferulic acid oo ka kooban oligosaccharides aashitada.
Gunaanad kale oo xiiso leh ayaa laga soo qaatay falanqaynta LC-MS ee jajabka oligosaccharide, sida ku cad Jaantuska S9 (hababka waxaa lagu arki karaa walxaha dheeraadka ah ee elektaroonigga ah). Jajabyada kooxaha hexose iyo -OAC ayaa si isdaba joog ah loo arkay intii lagu jiray isku-xidhka jajabka ACN-B. Natiijadani ma xaqiijinayso oo keliya kala-goynta lagu arkay falanqaynta glycome iyo MALDI-TOF, laakiin sidoo kale waxay bixisaa macluumaad cusub oo ku saabsan waxyaabaha ka soo baxa karbohaydraytka ee suurtagalka ah ee ku jira biomass-ka lignocellulosic ee hore loo daaweeyay.
Waxaan sidoo kale sameynay falanqayn ku saabsan halabuurka glycan ee jajabyada oligosaccharide annagoo adeegsanayna TMS glycan derivatization. Annagoo adeegsanayna GC-MS, waxaan go'aaminay isku-dhafka sonkorta neerfaha (aan ahayn kuwa la soo saaray) iyo kuwa aashitada leh (GluA iyo GalA) ee jajabka oligosaccharide (Jaantuska 5). Glucuronic acid waxaa laga helaa qaybaha aashitada leh ee C iyo D, halka galacturonic acid laga helo qaybaha aashitada leh ee A iyo B, kuwaas oo labaduba ah qaybo sare oo DP ah oo sonkor aashitada leh. Natiijooyinkani ma aha oo kaliya inay xaqiijinayaan xogtayada ELISA iyo MALDI, laakiin sidoo kale waxay la jaan qaadayaan daraasadihii hore ee ururinta oligosaccharide. Sidaa darteed, waxaan aaminsanahay in hababka difaaca jirka ee casriga ah ee isticmaalaya biotinylation ee oligosaccharides iyo baaritaanka ELISA ee xiga ay ku filan yihiin in lagu ogaado oligosaccharides-ka dib u milan ee muunado kala duwan oo bayooloji ah.
Maadaama hababka baaritaanka mAb ee ku salaysan ELISA lagu xaqiijiyay dhowr habab oo kala duwan, waxaan rabnay inaan si dheeraad ah u sahamino suurtagalnimada habkan cusub ee tirada. Laba oligosaccharides ganacsi, xylohexasaccharide oligosaccharide (XHE) iyo 23-α-L-arabinofuranosyl-xylotriose (A2XX), ayaa la iibsaday oo la tijaabiyay iyadoo la adeegsanayo hab cusub oo mAb ah oo lagu beegsanayo glycan-ka derbiga unugyada. Jaantuska 6 wuxuu muujinayaa isku xidhnaan toosan oo u dhexeeya calaamadda isku xidhka biotinylated iyo fiirsashada log ee fiirsashada oligosaccharide, taasoo soo jeedinaysa qaabka nuugista Langmuir ee suurtogalka ah. MAbs-ka dhexdooda, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, iyo CCRC-M151 waxay la xiriireen XHE, iyo CCRC-M108, CCRC-M109, iyo LM11 waxay la xiriireen A2XX oo u dhexeeya 1 nm ilaa 100 nano. Sababtoo ah helitaanka xaddidan ee unugyada difaaca jirka inta lagu jiro tijaabada, tijaabooyin xaddidan ayaa la sameeyay iyadoo la adeegsanayo fiirsashada oligosaccharide kasta. Waa in halkan lagu ogaadaa in qaar ka mid ah unugyada difaaca jirka ay si aad ah uga falceliyaan isla oligosaccharide-ka sida substrate-ka, maadaama ay ku xidhmaan epitopes-ka xoogaa kala duwan waxayna yeelan karaan isku xidhnaan aad u kala duwan. Hababka iyo saameynta aqoonsiga epitope-ka saxda ah waxay noqon doonaan kuwo aad u adag marka habka cusub ee mAb lagu dabaqo muunado dhab ah.
Laba oligosaccharides ganacsi ayaa loo isticmaalay si loo go'aamiyo kala duwanaanshaha ogaanshaha mAbs-yada kala duwan ee bartilmaameedsanaya glycan. Halkan, isku xirnaanta toosan ee fiirsashada log-ka ee fiirsashada oligosaccharides waxay muujinaysaa qaababka nuugista Langmuir ee (A) XHE oo leh mAb iyo (B) A2XX oo leh mAb. Epitopes-yada u dhigma waxay muujinayaan qaab-dhismeedka oligosaccharides-ka ganacsiga ee loo isticmaalo substrate-ka tijaabada.
Isticmaalka unugyada difaaca jirka ee monoclonal-ka ah ee bartilmaameedsanaya glycan (falanqaynta glycocomic ama baaritaanka mAb ee ku salaysan ELISA) waa qalab awood badan oo si qoto dheer loogu garto inta badan glycan-yada derbiga unugyada ee waaweyn ee sameeya biomass-ka dhirta. Si kastaba ha ahaatee, falanqaynta glycan-ka caadiga ah waxay sifaysaa oo keliya glycan-yada derbiga unugyada ee waaweyn, maadaama inta badan oligosaccharides aan si hufan loogu dhaqmin taarikada ELISA. Daraasaddan, shooladda galleyda ee AFEX-ka ah ee hore loo daweeyay waxaa si enzymatic ah loogu shubay biyo-xireen heer sare ah oo ku jira adkeysi badan. Falanqaynta sonkorta ayaa loo isticmaalay in lagu go'aamiyo isku-dhafka karbohaydraytyada derbiga unugyada ee dib-u-cusboonaysiinta ee ku jira hydrolyzate. Si kastaba ha ahaatee, falanqaynta mAb ee oligosaccharides-ka yar yar ee ku jira hydrolysates waa la dhayalsaday, waxaana loo baahan yahay qalab dheeraad ah si wax ku ool ah loogu joojiyo oligosaccharides-ka saxanadaha ELISA.
Waxaan halkan ku soo sheegaynaa hab cusub oo wax ku ool ah oo lagu joojinayo oligosaccharide si loo baaro mAb iyadoo la isku darayo biotinylation-ka oligosaccharide oo ay ku xigto baaritaanka ELISA ee saxannada dahaarka leh ee NeutrAvidin™. Oligosaccharides-ka biotinylated ee aan dhaqdhaqaaqa lahayn waxay muujiyeen isku xirnaan ku filan oo ku filan antibody-ga si loo suurtogeliyo ogaanshaha degdega ah iyo hufnaanta leh ee oligosaccharides-ka dib-u-cusboonaysiinta. Falanqaynta halabuurka oligosaccharides-kan adag ee ku salaysan spectrometry-ga tirada ayaa xaqiijisay natiijooyinka habkan cusub ee baaritaanka difaaca jirka. Sidaa darteed, daraasadahani waxay muujinayaan in isku-darka biotinylation-ka oligosaccharide iyo baaritaanka ELISA ee unugyada difaaca jirka ee monoclonal-ka ee glycan-ku bartilmaameedsan karo loo isticmaali karo in lagu ogaado isku-xirnaanta oligosaccharides-ka waxaana si weyn loogu dabaqi karaa daraasadaha kale ee bayoolaji ee qeexaya qaab-dhismeedka oligosaccharides.
Habkan ku salaysan biotin ee glycan profiling waa warbixinta ugu horreysa ee awood u leh inay baarto isku-xidhka karbohaydraytka ee oligosaccharides-ka milma ee ku jira biomass-ka dhirta. Tani waxay kaa caawinaysaa inaad fahamto sababta qaybo ka mid ah biomass-ku ay aad u adag yihiin marka ay timaado soo saarista shidaalka bio. Habkani wuxuu buuxinayaa farqiga muhiimka ah ee hababka falanqaynta glycome wuxuuna ku fidsan yahay codsigiisa noocyo badan oo substrates ah oo ka baxsan oligosaccharides-ka dhirta. Mustaqbalka, waxaan u isticmaali karnaa robotics-ka biotinylation-ka waxaanan u isticmaali karnaa habka aan u soo saarnay falanqaynta wax-soo-saarka sare ee muunadaha iyadoo la adeegsanayo ELISA.
Cawska galleyda (CS) ee laga beeray iniinyaha isku-dhafka ah ee Pioneer 33A14 ayaa laga soo goostay 2010 Kramer Farms oo ku taal Ray, Colorado. Iyadoo la raacayo ogolaanshaha milkiilaha dhulka, bayoolajiyadan waxaa loo isticmaali karaa cilmi-baaris. Shaybaarrada waxaa lagu kaydiyay qoyaan qalalan oo ka yar 6% bacaha lagu xidho sibka heerkulka qolka. Shaybaarrada waxaa lagu kaydiyay qoyaan qalalan oo ka yar 6% bacaha lagu xidho sibka heerkulka qolka. Образцы хранились сухими при влажности < 6% в пакетах Muunado waxaa lagu kaydiyay meel qalalan oo qoyaankeedu ka yar yahay 6% bacaha sibiyaha lagu dhejiyay heerkulka qolka.样品在室温下以干燥< 6% 的水分储存在自封袋中。样品在室温下以干燥< 6% Образцы хранят в пакетах с застежкой-молнией при комнатной температуре с влажностью <6%. Shaybaarrada waxaa lagu kaydiyaa bacaha sibka heerkulka qolka iyadoo qoyaanku ka yar yahay 6%.Daraasaddu waxay u hoggaansantay tilmaamaha maxalliga ah iyo kuwa qaranka. Falanqaynta isku-dhafka waxaa lagu sameeyay iyadoo la adeegsanayo hab-raaca NREL. Halabuurka waxaa la ogaaday inuu ka kooban yahay 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% borotiin iyo 13.4% dambas.
Cellic® CTec2 (138 mg borotiin/ml, VCNI 0001) waa isku-darka isku-dhafan ee cellulase, β-glucosidase iyo Cellic® HTec2 (157 mg borotiin/ml, lot VHN00001) oo laga keenay Novozymes (Franklinton, NC, USA)). Multifect Pectinase® (72 mg borotiin/mL), oo ah isku-darka isku-dhafan ee enzymes-ka wax u dhimaya pectin, waxaa ku deeqay DuPont Industrial Biosciences (Palo Alto, CA, USA). Fiirsashada borotiinka ensaymka waxaa lagu go'aamiyay iyadoo la qiyaasayo waxa ku jira borotiinka (iyo laga jarayo ka qayb qaadashada nitrogen-ka aan borotiinka ahayn) iyadoo la adeegsanayo falanqaynta nitrogen-ka Kjeldahl (habka AOAC 2001.11, Dairy One Cooperative Inc., Ithaca, NY, USA). Diatomaceous earth 545 waxaa laga soo iibsaday EMD Millipore (Billerica, MA). Kaarboon firfircoon (DARCO, 100 granules mesh), Avicel (PH-101), beech xylan, iyo dhammaan kiimikooyinka kale waxaa laga soo iibsaday Sigma-Aldrich (St. Louis, MO).
Daawaynta hore ee AFEX waxaa lagu sameeyay GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA). Daawaynta hore waxaa lagu sameeyay heerkul ah 140° C. muddo 15 daqiiqo ah. 46 waqti joogitaan ah oo ah saamiga 1:1 ee ammonia aan biyo lahayn iyo biomass oo ah 60% (w/w) oo lagu shubay fal-galiye bir ah oo aan miridh lahayn (Parr Instruments Company). Waxay qaadatay 30 daqiiqo. Fal-galiyaha waxaa la gaarsiiyay 140°C waxaana ammonia si degdeg ah loo sii daayay, taasoo u oggolaanaysa bayomasku inuu si dhakhso ah ugu soo laabto heerkulka qolka. Halabuurka shooladda galleyda ee hore loo daweeyay ee AFEX (ACS) waxay la mid ahayd tan shooladda galleyda ee aan la daweyn (UT-CS).
Adkaha sare ee ACSH 25% (w/w) (qiyaastii 8% dextran load) ayaa loo diyaariyey sidii agab bilow ah oo loogu talagalay wax soo saarka baaxadda weyn ee oligosaccharides. Enzymatic hydrolysis ee ACS waxaa lagu sameeyay iyadoo la adeegsanayo isku darka ensaymka ganacsiga oo ay ku jiraan Cellic® Ctec2 10 mg protein/g glucan (biomass-ka hore loo daaweeyay), Htec2 (Novozymes, Franklinton, NC), 5 mg protein/g glucan, iyo Multifect Pectinase (Genencor Inc, USA). ), 5 mg protein/g dextran. Enzymatic hydrolysis waxaa lagu sameeyay bioreactor 5-litir ah oo leh mug shaqo oo ah 3 litir, pH 4.8, 50°C iyo 250 rpm. Ka dib hydrolysis muddo 96 saacadood ah, hydrolyzate waxaa lagu soo ururiyay centrifugation 6000 rpm muddo 30 daqiiqo ah ka dibna 14000 rpm muddo 30 daqiiqo ah si looga saaro adkeysiga aan la hydrolyzed. Dabadeedna waxaa lagu shubay biyo nadiif ah iyadoo la adeegsanayo weel shaandho ah oo cabirkiisu yahay 0.22 mm. Biyaha la sifeeyay waxaa lagu kaydiyay dhalooyin nadiif ah heerkul ah 4°C ka dibna waxaa lagu kala qaybiyay kaarboon.
Falanqaynta isku-darka muunadaha bayoomaaska ku salaysan soosaarka iyadoo la raacayo hababka falanqaynta shaybaarka NREL: diyaarinta muunadaha falanqaynta isku-dhafka (NREL/TP-510-42620) iyo go'aaminta karbohaydraytyada qaab-dhismeedka iyo lignin ee bayoomaaska (NREL/TP-510 - 42618)47.
Falanqaynta oligosaccharide ee durdurka hydrolyzate waxaa lagu sameeyay miisaan 2 ml ah iyadoo la adeegsanayo habka hydrolysis-ka acid-ka ku salaysan autoclave. Ku qas muunadda hydrolyzate 69.7 µl oo ah 72% sulfuric acid tuubo dhaqan oo dabool leh oo 10 ml ah oo ku dhex shiil 1 saac oo ku dul shiil miis 121 °C ah, ku qabooji baraf oo ku shaandhee dhalo chromatography dareere ah oo waxqabad sare leh (HPLC). Isugeynta oligosaccharides waxaa lagu go'aamiyay iyadoo laga jarayo isku-darka monosaccharides ee muunadda aan hydrolyzate ahayn wadarta guud ee isku-darka sonkorta ee muunadda lagu hydrolyzate-ka.
Fiirsashada gulukooska, xylose, iyo arabinose ee ku jira biomass-ka aashitada lagu shubay ayaa la falanqeeyay iyadoo la adeegsanayo nidaamka Shimadzu HPLC oo ku qalabaysan autosampler, kuleyliyaha tiirka, bamka isocratic, iyo qalabka ogaanshaha tusmada refractive ee ku yaal tiirka Bio-Rad Aminex HPX-87H. Tiirka waxaa lagu hayay 50°C waxaana lagu daboolay 0.6 ml/daqiiqo 5 mM H2SO4 oo biyo ah. socodka.
Subagga hydrolyzate-ka ayaa la qasi jiray oo la falanqeeyay si loo ogaado heerka monomer iyo oligosaccharide. Sonkorta monomeric-ga ah ee la helay ka dib markii la shubay enzymatic hydrolysis waxaa falanqeeyay HPLC oo ku qalabaysan tiir Bio-Rad (Hercules, CA) Aminex HPX-87P ah iyo tiir ilaaliye dambas ah. Heerkulka tiirka waxaa lagu hayay 80°C, biyaha waxaa loo isticmaalay marxaladda guurguurta iyadoo heerka socodka uu yahay 0.6 ml/daqiiqo. Oligosaccharides-ka waxaa lagu go'aamiyay hydrolysis aashitada khafiifka ah 121°C sida ku cad hababka lagu sharraxay tixraaca 41, 48, 49.
Falanqaynta Saccharide waxaa lagu sameeyay haraaga bayoolajiga cayriin, AFEX oo hore loo daaweeyay iyo dhammaan haraaga bayoolajiga aan la biyo-siinin (oo ay ku jiraan soo saarista soo saarista derbiga unugyada taxanaha ah iyo baaritaanka mAb-kooda) iyadoo la adeegsanayo hababka hore loo sharraxay 27, 43, 50, 51. Falanqaynta glycome-ka, haraaga aan milmin aalkolada ee walaxda derbiga unugyada dhirta waxaa laga diyaariyey haraaga bayoolajiga waxaana loo mariyey soo saarid taxane ah iyadoo la adeegsanayo reagents-ka sii kordhaya ee gardarrada leh sida ammonium oxalate (50 mM), sodium carbonate (50 mM iyo 0.5% w/v), CON. (1M iyo 4M, labaduba waxay leeyihiin 1% w/v sodium borohydride) iyo acid chlorite sida hore loogu sharraxay52,53. Soo saarista waxaa markaa lagu sameeyay ELISA iyadoo la adeegsanayo guddi adag oo mAb50s ah oo loo jiheeyay glycan-ka derbiga unugyada, falcelinta isku xidhka mAb-na waxaa loo soo bandhigay khariidad kuleyl. mAbs oo bartilmaameedsanaya glycan-ka derbiga unugyada dhirta waxaa laga soo iibsaday kaydka shaybaarka (CCRC, taxanaha JIM iyo MAC).
Biyotinylation hal tallaabo ah oo oligosaccharides ah. Isku-darka karbohaydraytyada leh biotin-LC-hydrazide waxaa lagu sameeyay iyadoo la adeegsanayo habka soo socda. Biotin-LC-hydrazide (4.6 mg/12 μmol) waxaa lagu milmay dimethyl sulfoxide (DMSO, 70 μl) iyadoo si xooggan loo walaaqayo oo lagu kululeynayo 65° C. muddo 1 daqiiqo ah. Glacial acetic acid (30 µl) ayaa lagu daray isku-darkana waxaa lagu shubay sodium cyanoborohydride (6.4 mg/100 µmol) waxaana gebi ahaanba la milmay ka dib markii lagu kululeeyay 65° C. muddo 1 daqiiqo ah. Kadib, laga bilaabo 5 ilaa 8 μl oo isku-darka falgalka ah ayaa lagu daray oligosaccharide qalalan (1-100 nmol) si loo helo 10-laab ama ka badan xad-dhaafka molar ee calaamadda dhammaadka yareynaya. Falcelinta waxaa lagu sameeyay 65°C muddo 2 saacadood ah, ka dibna muunado ayaa isla markiiba la nadiifiyay. Ma jirin sodium cyanoborohydride lagu isticmaalay tijaabooyinka calaamadaynta iyada oo aan la dhimin, muunado-na waxaa lagu falceliyay heerkul ah 65° C. muddo 2.5 saacadood ah.
Dahaarka ELISA iyo dhaqidda muunadaha oligosaccharides-ka biotinylated. 25 μl oo muunado bayotinylated ah (100 μl oo muunad kasta oo la ururiyay oo lagu qasi jiray 5 ml oo ah 0.1 M Tris buffer (TBS)) ayaa lagu daray ceel kasta oo saxanka dahaaran ee avidin. Ceelasha xakamaynta waxaa lagu dahaadhay 50 μl oo biotin ah oo leh isku-darka 10 μg/ml oo ah 0.1 M TBS. Biyo la baabi'iyay ayaa loo isticmaalay dahaar ahaan cabbiraadaha madhan. Kiniinka waxaa lagu kululeeyay 2 saacadood heerkulka qolka mugdiga ah. Saxanka ku dhaq 3 jeer caano 0.1% ah oo la shiiday oo ku jira 0.1 M TBS iyadoo la adeegsanayo barnaamijka lambarka 11 ee Grenier flat 3A.
Ku darista iyo dhaqidda unugyada difaaca jirka ee aasaasiga ah. Ku dar 40 µl oo ah unugyada difaaca jirka ee aasaasiga ah ceel kasta. Ku shid saxanka microplate-ka 1 saac heerkulka qolka mugdiga ah. Kadib saxanadaha waxaa lagu dhaqay 3 jeer caano 0.1% ah 0.1M TBS iyadoo la isticmaalayo barnaamijka dhaqidda #11 ee Grenier Flat 3A.
Ku dar antibody labaad oo dhaq. Ku dar 50 µl oo antibody labaad oo jiirka/jiirka ah (oo lagu qasi jiray 1:5000 oo caano ah 0.1% oo ku jira 0.1 M TBS) ceel kasta. Ku shid microplate-ka 1 saac heerkulka qolka mugdiga ah. Microplate-yada waxaa markaa lagu dhaqay 5 jeer oo lagu dhaqay caano 0.1% oo ku jira 0.1 M TBS iyadoo la adeegsanayo barnaamijka dhaqidda saxanka Grenier Flat 5A #12.
Ku darista substrate. Ku dar 50 µl oo ah 3,3′, 5,5′-tetramethylbenzidine (TMB) substrate-ka salka (adigoo ku daraya 2 dhibcood oo buffer ah, 3 dhibcood oo TMB ah, 2 dhibcood oo hydrogen peroxide ah 15 ml oo biyo ah oo la miiray). Diyaari substrate-ka TMB. oo rogrog ka hor intaadan isticmaalin). Ku shid saxanka heerkulka qolka 30 daqiiqo. Mugdiga.
Dhammaystir tallaabada oo akhri kiniinka. Ku dar 50 µl oo ah 1 N sulfuric acid ceel kasta oo diiwaan geli nuugista laga bilaabo 450 ilaa 655 nm adoo isticmaalaya akhristaha ELISA.
Diyaari xalal 1 mg/ml ah oo ka mid ah falanqeeyayaashan biyaha la kala diray: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine. (glcNAc), N-acetylgalactosamine (galNAc), inositol (heerka gudaha). Laba heer ayaa la diyaariyey iyadoo lagu darayo xalalka sonkorta 1 mg/mL ee lagu muujiyay Shaxda 1. Muunadaha waa la qaboojiyaa oo la lyophilides ku shubaa -80° C. ilaa dhammaan biyaha laga saaro (badanaa qiyaastii 12-18 saacadood).
Ku dar 100–500 µg oo muunad ah si aad ugu xidho tuubooyinka daboolka dheelitir falanqayn ah. Diiwaangeli xaddiga lagu daray. Waxaa ugu wanaagsan in muunadda lagu milmo xoog gaar ah oo dareere ah oo lagu daro tuubada iyadoo loo eegayo dareere ahaan. Isticmaal 20 µl oo ah 1 mg/ml inositol oo ah halbeeg gudaha ah tuubo kasta oo muunad ah. Cadadka halbeegga gudaha ee lagu daray muunadda waa inuu la mid ahaadaa cadadka halbeegga gudaha ee lagu daray tuubada caadiga ah.
Ku dar 8 ml oo methanol aan biyo lahayn dhalada daboolka boolal. Kadibna 4 ml oo ah 3 N. methanolic HCl oo dareere ah, oo daboolan oo la ruxruxo. Habkani ma isticmaalo biyo.
Ku dar 500 µl oo ah 1 M HCl methanol xal ah muunadaha oligosaccharide iyo tuubooyinka TMS ee caadiga ah. Muunadaha waxaa lagu kululeeyay habeenkii (168 saacadood) heerkul ah 80° C. baloog kuleyl ah. Ku qallaji badeecada methanolysis heerkulka qolka adoo isticmaalaya manifold qalajin ah. Ku dar 200 µl MeOH oo mar kale qalaji. Habkan waxaa lagu celceliyaa laba jeer. Ku dar 200 µl oo methanol ah, 100 µl oo pyridine ah iyo 100 µl oo acetic anhydride ah muunadda oo si fiican isku walaaq. Muunadaha ku rid heerkulka qolka muddo 30 daqiiqo ah. oo qalaji. Ku dar 200 µl oo methanol ah oo mar kale qalaji.
Ku dar 200 µl oo Tri-Sil ah oo ku kululee tuubada daboolan 20 daqiiqo. 80°C, ka dibna ku qabooji heerkulka qolka. Isticmaal qalab qalajin ah si aad u sii qalajiso muunadda ilaa mugga qiyaastii 50 µl. Waa muhiim in la ogaado inaanan u oggolaan in muunaduhu si buuxda u qalalaan.
Ku dar 2 ml oo hexane ah oo si fiican isku walaaq adigoo ku shubaya. Cidhifyada Pasteur pipettes (5-8 mm) ku buuxi gabal dhogor galaas ah adigoo dhogorta galaaska dul saaraya tuubo dhexroorkeedu yahay 5-3/4 inji. Muunadaha waxaa lagu centrifuge gareeyay 3000 g muddo 2 daqiiqo ah. Wixii haraaga aan milmi karin waa la soo daadinayaa. Muunadda ku qallaji 100-150 µl. Mug qiyaastii 1 μl ah ayaa lagu duray GC-MS heerkulkii ugu horreeyay ee ahaa 80 °C iyo waqtigii ugu horreeyay oo ahaa 2.0 daqiiqo (Jadwalka 2).