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Uburyo bushya bwo gukingira no gukwirakwiza sprometrike yuburyo bwo gusesengura byimazeyo oligosaccharide idahwema kubika ibigori byateguwe na AFEX.Lignocellulosic biomass nubundi buryo burambye bwibicanwa bya fosile kandi bikoreshwa cyane mugutezimbere ibinyabuzima bigamije gukora ibicuruzwa nkibiryo, ibiryo, lisansi na chimique.Urufunguzo rw'ikoranabuhanga ni ugutezimbere uburyo bwo guhatanira ibiciro kugirango uhindure karubone nziza igaragara mu rukuta rw'utugingo ngengabuzima mu isukari yoroshye nka glucose, xylose na arabinose.Kubera ko biomass ya lignocellulosique yinangiye cyane, igomba gukorerwa imiti yubushyuhe (urugero, ammonia fibre exfoliation (AFEX), acide acide (DA), amavuta ya ionic (IL)) hamwe nubuvuzi bwibinyabuzima (urugero, hydrolysis enzymatique na fermentation ya mikorobe) hamwe kugirango ibone ibicuruzwa byifuzwa..Nyamara, iyo imisemburo ya fungal yubucuruzi ikoreshwa mugikorwa cya hydrolysis, 75-85% gusa byisukari yumuti yashizwemo ni monosaccharide, naho 15-25% isigaye irashonga, oligosaccharide idashobora gukoreshwa, idahora iboneka mikorobe.Mbere, twatandukanije neza kandi dusukuye ibishishwa byinangiye oligosaccharide dukoresheje guhuza karubone na diatomaceous isi itandukanya hamwe nubunini bwa chromatografiya, kandi tunakora iperereza kumiterere yabyo.Twabonye ko oligosaccharide irimo urwego rwo hejuru rwa polymerisiyonike (DP) methylated uronic acide acide iragoye gutunganya hamwe na enzyme yubucuruzi ivanze kuruta DP nkeya na oligosaccharide idafite aho ibogamiye.Hano turatanga raporo yo gukoresha uburyo butandukanye bwinyongera, harimo gushushanya glycan ukoresheje antibodiyite za monoclonal (mAbs) zihariye ziterwa na biomass glycans kugirango ziranga imigozi ya glycan mu nkuta z’ibimera hamwe na hydrolysates ya enzymatique, matrix ifashwa na laser desorption ionisiyoneri, igihe-cyo guhaguruka-mass-spectrometry..MALDI-TOF-MS) ikoresha imiterere-yamakuru yo kwisuzumisha yabonetse na spekitroscopi nyuma yo kwangirika kwa kabiri kwa ion mbi, gazi chromatografiya hamwe na mass spectrometrie (GC-MS) kugirango iranga isano ya oligosaccharide hamwe na derivatisation.Bitewe nubunini buke bwa oligosaccharide (DP 4–20), izo molekile ziragoye gukoresha mAb guhuza no kuranga.Kugira ngo iki kibazo gikemuke, twakoresheje uburyo bushya bwa biotin conjugation bushingiye kuri oligosaccharide immobilisation yerekana neza umubare munini wa DP nkeya ya oligosaccharide ya microplate hejuru, hanyuma igakoreshwa muri sisitemu yo hejuru ya mAb kugirango isesengure ryihariye.Ubu buryo bushya buzorohereza iterambere ryiterambere ryinshi ryinjiza glycome assays mugihe kizaza gishobora gukoreshwa mugutandukanya no kuranga oligosaccharide igaragara muri biomarkers mugamije gusuzuma.
Lignocellulosic biomass, igizwe n’ubuhinzi, amashyamba, ibyatsi n’ibiti by’ibiti, ni amatungo ashobora gutunganyirizwa mu bicuruzwa bikomoka ku binyabuzima, birimo ibiryo, ibiryo, lisansi n’ibikoresho bya chimique kugira ngo bitange umusaruro w’agaciro1.Carbohydrates (nka selile na hemicellulose) iboneka mu rukuta rw'utugingo ngengabuzima twa depolymerized muri monosaccharide hakoreshejwe gutunganya imiti no guhindura ibinyabuzima (nka hydrolysis enzymatique na fermentation ya mikorobe).Ibisanzwe mbere yo kuvura harimo kwagura fibre fibre ammonia (AFEX), acide dilute (DA), ionic fluid (IL), hamwe no guturika kwamazi (SE), ikoresha imiti nubushyuhe kugirango igabanye umusaruro wa lignocellulose ufungura inkuta zi selile3,4.kunangira ibintu, 5. Hydrolysis ya Enzymatique ikorerwa ku mutwaro uremereye ukoresheje ubucuruzi bukora karbone-hydrata irimo imisemburo (CAZymes) hamwe na fermentation ya mikorobe ikoresheje imisemburo ya transgenji cyangwa bagiteri kugirango ikore ibicanwa bishingiye kuri bio na chimique 6.
CAZymes mumisemburo yubucuruzi igizwe nuruvange rwimvange rwimisemburo ihuza imbaraga za karubone-hydrata-isukari ikora kugirango ibe monosaccharide2,7.Nkuko twabibabwiye kare, urusobe rugoye rwa polimeri ya aromatic ya lignine hamwe na karubone ya hydrata ituma idashobora gukoreshwa cyane, bigatuma habaho isukari ituzuye, ikusanya 15-25% byimibonano mpuzabitsina oligosaccharide idakorwa mugihe cya hydrolysis ya enzymatique ya biomass yabigenewe.Iki nikibazo gisanzwe hamwe nuburyo butandukanye bwo kwitegura biomass.Impamvu zimwe zitera iki cyuho zirimo kubuza enzyme mugihe cya hydrolysis, cyangwa kubura cyangwa urwego ruke rwimisemburo yingenzi isabwa guca isukari muri biomass yibihingwa.Gusobanukirwa imiterere nibiranga isukari, nkisukari yisukari muri oligosaccharide, bizadufasha kunoza ihinduka ryisukari mugihe cya hydrolysis, bigatuma inzira ya biotechnologie ihenze-hamwe nibikomoka kuri peteroli.
Kumenya imiterere ya karubone nziza biragoye kandi bisaba guhuza uburyo nka chromatografiya y'amazi (LC) 11,12, magnetic magnetic resonance spectroscopy (NMR) 13, capillary electrophoresis (CE) 14,15,16 na mass spectrometrie (MS) 17., cumi n'umunani.Uburyo bwa MS nkigihe-cyo-guhaguruka-mass mass spectrometrie hamwe na laser desorption hamwe na ionisation ukoresheje matrix (MALDI-TOF-MS) nuburyo butandukanye bwo kumenya imiterere ya karubone.Vuba aha, Gutandukana-Guterwa no Gutandukana (CID) tandem MS ya sodium ion yongeweho yakoreshejwe cyane kugirango hamenyekane urutoki rujyanye na oligosaccharide ifatanye, ibishushanyo bya anomeric, uko bikurikirana, hamwe nishami rya 20, 21.
Isesengura rya Glycan nigikoresho cyiza cyo kumenya byimbitse imiterere ya karubone.Ubu buryo bukoresha antibodiyite za monoclonal (mAbs) zerekeza ku gutera urukuta rwa selile glycan nk'ubushakashatsi bwo gusobanukirwa isano ya karubone nziza.MAbs zirenga 250 ziraboneka kwisi yose, zakozwe zirwanya umurongo utandukanye wa oligosaccharide ukoresheje amashami atandukanye24.MAbs nyinshi zagiye zikoreshwa cyane mu kuranga imiterere, ibigize, no guhindura urukuta rw'utugingo ngengabuzima, kuko hari itandukaniro rikomeye bitewe n'ubwoko bw'utugingo ngengabuzima, urugingo, imyaka, icyiciro cy'iterambere, n'ibidukikije bikura 25,26.Vuba aha, ubu buryo bwakoreshejwe mugusobanukirwa abaturage ba viticle muri sisitemu y’ibimera n’inyamaswa n’uruhare rwabo mu bwikorezi bwa glycan nkuko byagenwe n’ibimenyetso bito, ibyiciro byiterambere, cyangwa ibidukikije, no kumenya ibikorwa byimisemburo.Bimwe mubikorwa bitandukanye bya glycans na xylans byagaragaye harimo pectine (P), xylan (X), mannan (M), xyloglucans (XylG), glucans ivanze (MLG), arabinoxylan (ArbX), galactomannan (GalG), aside glucuronic-arabinoxylan (GArbXan) na arabino-galactan (GArbX).
Nubwo, nubwo hashyizweho ingufu zose zubushakashatsi, ubushakashatsi buke bwibanze ku miterere yo kwirundanya kwa oligosaccharide mugihe cya hydrolysis itwara ibintu byinshi (HSL), harimo kurekura oligosaccharide, uburebure bwa oligomerique ihinduka mugihe cya hydrolysis, polymers zitandukanye za DP, hamwe nu murongo.kugabura 30,31,32.Hagati aho, nubwo isesengura rya glycan ryerekanye ko ari igikoresho cyingirakamaro mu gusesengura byimazeyo imiterere ya glycan, biragoye gusuzuma amazi ya elegitoronike ya DP oligosaccharide ukoresheje uburyo bwa antibody.Gitoya DP oligosaccharide ifite uburemere bwa molekuline iri munsi ya 5-10 kDa ntabwo ihuza plaque ya ELISA 33, 34 kandi irakaraba mbere yo kongeramo antibody.
Hano, kunshuro yambere, twerekanye ELISA isuzuma ryamasahani yometse kuri avidin dukoresheje antibodiyite za monoclonal, duhuza inzira imwe ya biotinylation yuburyo bumwe bwo gukonjesha oligosaccharide yangirika hamwe nisesengura rya glycome.Uburyo bwacu bwo gusesengura glycome bwemejwe na MALDI-TOF-MS na GC-MS ishingiye ku isesengura ryuzuzanya rya oligosaccharide dukoresheje trimethylsilyl (TMS) ikomoka ku isukari ya hydrolyzed.Ubu buryo bushya bushobora gutezwa imbere nkuburyo bwinjiza cyane mugihe kizaza kandi ugashaka uburyo bwagutse mubushakashatsi bwibinyabuzima35.
Guhindura nyuma yubusobanuro bwa enzymes na antibodies, nka glycosylation, 36 bigira ingaruka kubikorwa byabo byibinyabuzima.Kurugero, impinduka muri glycosylation ya proteine ​​za serumu zigira uruhare runini muri arthritis inflammatory, kandi impinduka za glycosylation zikoreshwa nkibimenyetso byo gusuzuma37.Indwara ya glycans zitandukanye yagiye ivugwa mu bitabo kugira ngo igaragare byoroshye mu ndwara zitandukanye, zirimo n'indwara zidakira zandurira mu nzira zo mu gifu n'umwijima, kwandura virusi, intanga ngore, amabere, na kanseri ya prostate 38,39,40.Gusobanukirwa imiterere ya glycans ukoresheje antibody ishingiye kuri glycan ELISA bizatanga ibyiringiro byinyongera mugupima indwara udakoresheje uburyo bukomeye bwa MS.
Ubushakashatsi bwatubanjirije bwerekanye ko oligosakisaride yinangiye yagumye idafite amazi nyuma yo kwisuzumisha hamwe na hydrolysis ya enzymatique (Ishusho 1).Mubikorwa byacu byasohotse mbere, twateje imbere uburyo bwo gukuramo amakara akomeye kugirango akuremo oligosakisaride na AFEX-ibigori byateguwe na hydrolyzate (ACSH) 8.Nyuma yo gukuramo bwa mbere no gutandukana, oligosaccharide yarushijeho gucikamo ibice byerekana ubunini bwa chromatografiya (SEC) hanyuma ikusanywa hakurikijwe uburemere bwa molekile.Isukari ya monomers na oligomers yasohotse mubitekerezo bitandukanye byasesenguwe nisesengura ryibigize isukari.Iyo ugereranije ibirimo isukari oligomers yabonetse muburyo butandukanye bwo kwitegura, kuba oligosaccharide yinangiye ni ikibazo gikunze kugaragara muguhindura biomass na monosaccharide kandi bishobora gutuma igabanuka ryumusukari byibuze 10-15% ndetse bikagera kuri 18%.Amerika.Ubu buryo bukoreshwa mugukora umusaruro munini wibice bya oligosaccharide.Ibisubizo bya ACH hamwe nibice byakurikiyeho bifite uburemere butandukanye bwa molekile byakoreshejwe nkibikoresho byo kugerageza kuranga oligosakisaride muriyi mirimo.
Nyuma yo kwisuzumisha hamwe na hydrolysis ya enzymatique, oligosaccharide idahwema kuguma idafite amazi.Hano (A) ni uburyo bwo gutandukanya oligosaccharide aho oligosaccharide itandukanijwe na AFEX yateguwe na hydrolyzate y'ibigori ya AFEX (ACSH) ikoresheje uburiri bupakiye bwa karubone ikora cyane hamwe na diatomaceous;(B) Uburyo bwo gutandukanya oligosaccharide.Oligosaccharide yatandukanijwe nubunini bwo gutandukanya chromatografiya (SEC);.Imiterere ya hydrolysis ya Enzymatique: ibintu byinshi biremereye 25% (w / w) (hafi ya 8% yikuramo glucan), hydrolysis yamasaha 96, 20 mg / g enzyme yubucuruzi yipakurura (Ctec2: Htec2: MP-2: 1: 1)
Isesengura rya Glycan ryerekanye ko ari igikoresho cyingirakamaro mu isesengura ryuzuye ryimiterere ya glycans mu bice bitandukanijwe n’ibisigazwa bya biomass.Nyamara, isukari ya elegitoronike ya elegitoronike ntisobanurwa neza ukoresheje ubu buryo gakondo41 kuko uburemere buke bwa molekile oligosaccharide biragoye kwimuka ku byapa bya ELISA kandi bikogejwe mbere yo kongeramo antibody.Kubwibyo, kugirango antibody ihuze kandi iranga, uburyo bumwe bwa biotinylation bwakoreshejwe mukwambara amakariso, oligosaccharide idahuye na plaque ya ELISA.Ubu buryo bwageragejwe hakoreshejwe ACSH twabanje gukora hamwe nigice gishingiye ku buremere bwacyo (cyangwa urwego rwa polymerisation, DP).Intambwe imwe ya biotinylation yakoreshejwe mu kongera oligosaccharide ihuza ubumwe hongerwamo biotin-LC-hydrazide kugabanya impera ya karubone (Ishusho 2).Mu gukemura, itsinda rya hemiacetal kumpera yo kugabanuka ryitwara hamwe nitsinda rya hydrazide ya biotin-LC-hydrazide kugirango ibe umurunga wa hydrazone.Imbere ya agent igabanya NaCNBH3, imiyoboro ya hydrazone iragabanuka kubicuruzwa biotinilated bihamye.Hamwe no guhindura isukari igabanya impera, guhuza DP oligosaccharide nkeya kuri plaque ya ELISA byashobokaga, kandi mubushakashatsi bwacu ibi byakorewe kumasahani yometse kuri avidin ukoresheje mAbs yibasiwe na glycan.
Kwerekana antibodiyite za monoclonal zishingiye kuri ELISA kuri biotinylated oligosaccharide.Hano.
Isahani yometse kuri Avidin hamwe na antibodiyite ya oligosaccharide-conjugated noneho yongewe kuri antibodi yibanze nayisumbuye hanyuma yozwa muburyo bworoshye kandi bworoshye.Nyuma yo guhuza antibody irangiye, ongeramo TMB substrate kugirango ushireho isahani.Amaherezo reaction yahagaritswe na acide sulfurike.Isahani yubushakashatsi yasesenguwe hifashishijwe umusomyi wa ELISA kugirango hamenyekane imbaraga zihuza buri antibody kugirango hamenyekane antibody yihariye ihuza.Ushaka ibisobanuro birambuye hamwe nibipimo byubushakashatsi, reba igice kijyanye "Ibikoresho nuburyo".
Twerekana akamaro k'ubu buryo bushya bwatejwe imbere mubikorwa byihariye turanga oligosaccharide ya elegitoronike iboneka muri ACSH kimwe no mubice bya oligosaccharide bitavanze kandi bisukuye bitandukanijwe na hydrolysates ya lignocellulosique.Nkuko bigaragara ku gishushanyo cya 3, xylans isimburwa na epitope ikunze kugaragara muri ACSH ikoresheje uburyo bwa bioacylated glycome assay ni ubusanzwe ni uronic (U) cyangwa methyluronic (MeU) na arabinogalactans pectic.Benshi muribo wasangaga no mubushakashatsi bwatubanjirije ku isesengura rya glycans ya hydrolyzed solide (UHS) 43.
Kumenya epitopes ya oligosaccharide ya recalcitrant ukoresheje antibody ya monoclonal yerekeza kurukuta rwa selile glycan.Igice cya "kutagira aho kibogamiye" ni agace ka ACN naho agace ka "acide" nigice cya FA.Umutuku urabagirana kuri hotmap yerekana epitope iri hejuru, naho ubururu bwerurutse bwerekana inyuma.Ibara ryamabara kumunzani rishingiye kubiciro bya OD mbisi kubisobanuro N = 2.Epitope nyamukuru yamenyekanye na antibodies yerekanwa iburyo.
Izi nyubako zitari selile ntizishobora gutoborwa na selile ikunze kugaragara hamwe na hemicellulase munganda zivanze na enzyme yubucuruzi yapimwe, ikubiyemo imisemburo yubucuruzi ikoreshwa cyane.Kubwibyo, enzymes nshya zifasha zirakenewe kuri hydrolysis.Hatariho imisemburo ikenewe itari selile, iyi sano idafite selile irinda ihinduka ryuzuye kuri monosaccharide, kabone niyo polimeri yababyeyi yabyo isukuye cyane mubice bigufi hanyuma bigashonga hakoreshejwe imvange ya enzyme yubucuruzi.
Ubundi bushakashatsi bwakozwe ku gukwirakwiza ibimenyetso n'imbaraga zacyo bwerekanaga ko epitopa ihuza yari hasi mu bice byinshi by'isukari ya DP (A, B, C, DP kugeza kuri 20+) ugereranije no mu bice bike bya DP (D, E, F, DP) mu bipimo) (Ishusho 1).Ibice bya acide bikunze kugaragara muri epitopes itari selile kuruta ibice bitabogamye.Ibi bintu bihuye nuburyo bwagaragaye mubushakashatsi bwatubanjirije, aho DP hamwe na acide nyinshi ya aside irwanya hydrolysis enzymatique.Kubwibyo, kuba epitopa ya glycan itari selile na insimburangingo U na MeU zirashobora kugira uruhare runini mugutuza kwa oligosaccharide.Twabibutsa ko guhuza no gutahura neza bishobora kuba ikibazo kuri DP oligosaccharide nkeya, cyane cyane iyo epitope ari dimeric cyangwa trimeric oligosaccharide.Ibi birashobora kugeragezwa ukoresheje oligosaccharide yubucuruzi yuburebure butandukanye, buri kimwe kirimo epitope imwe gusa ihuza mAb runaka.
Rero, ikoreshwa rya antibodiyite zihariye zagaragaje ubwoko bumwe bwimigozi isubirwamo.Bitewe n'ubwoko bwa antibody yakoreshejwe, uburyo bukwiye bwo guhuza, hamwe n'imbaraga z'ikimenyetso itanga (byinshi kandi bike cyane), imisemburo mishya irashobora kumenyekana hanyuma ikongerwaho igice-cyinshi muburyo bwa enzyme ivanze na glycoconversion yuzuye.Dufashe isesengura rya ACSH oligosaccharide nkurugero, turashobora gukora base de base ya glycan kuri buri kintu cya biomass.Hano twakwibutsa ko isano itandukanye ya antibodies igomba kwitabwaho, kandi niba isano yabo itazwi, ibi bizatera ingorane zimwe mugihe ugereranije ibimenyetso bya antibodi zitandukanye.Byongeye kandi, kugereranya imigozi ya glycan irashobora gukora neza hagati yintangarugero kuri antibody imwe.Izi nkunga zinangiye zirashobora guhuzwa noneho nububiko bwa CAZyme, aho dushobora gutangariza imisemburo, guhitamo enzymes zabakandida no kugerageza imisemburo isenya inkwano, cyangwa guteza imbere mikorobe kugirango tugaragaze iyo misemburo yo gukoresha muri biorefineries44.
Kugirango dusuzume uburyo uburyo bwo gukingira bwuzuza ubundi buryo bwo kuranga uburemere buke bwa oligosaccharide igaragara muri hydrolysate ya lignocellulosique, twakoze MALDI (Igishusho 4, S1-S8) no gusesengura sakaride ikomoka kuri TMS ishingiye kuri GC-MS ku gice kimwe (Igicapo 5) igice cya oligosaccharide.MALDI ikoreshwa mukugereranya niba ikwirakwizwa ryinshi rya molekile ya oligosaccharide ihuye nuburyo bugenewe.Ku mutini.4 yerekana MC yibice bitagira aho bibogamiye ACN-A na ACN-B.ACN-Isesengura ryemeje isukari ya pentose itandukanye kuva DP 4–8 (Igishusho 4) kugeza DP 22 (Ishusho S1), uburemere bwayo bukaba buhuye na MeU-xylan oligosaccharide.Isesengura rya ACN-B ryemeje urukurikirane rwa pentose na glucoxylan hamwe na DP 8-15.Mubikoresho byinyongera nkibishushanyo S3, amakarita yo gukwirakwiza misa ya FA-C yerekana ikarita yerekana (Me) U isimbuye isukari ya pentose hamwe na DP ya 8-15 ihuza na xylans yasimbuwe iboneka muri ELISA ishingiye kuri mAb.Ibice birahoraho.
MALDI-MS sprifike ya soluble idakurikiza oligosaccharide igaragara muri ACS.Hano,.
Isesengura ryibigize ibisigazwa bya glycan bya oligosaccharide yangiritse.Hano (A) TMS sakaride igizwe nibice bitandukanye bya oligosaccharide yabonetse ukoresheje isesengura rya GC-MS.(B) Imiterere yisukari itandukanye ikomoka kuri TMS iboneka muri oligosaccharide.ACN - agace ka acetonitrile karimo oligosakisaride idafite aho ibogamiye na FA - agace ka ferulic aside irimo aside oligosaccharide.
Undi mwanzuro ushimishije wavuye mu isesengura rya LC-MS ryerekeye agace ka oligosaccharide, nkuko bigaragara ku gishushanyo S9 (uburyo bushobora kugaragara mubikoresho byiyongera kuri elegitoroniki).Ibice by'itsinda rya hexose na -OAc byagaragaye inshuro nyinshi mugihe cyo guhuza agace ka ACN-B.Ubu bushakashatsi ntabwo bwemeza gusa gucikamo ibice byagaragaye mu isesengura rya glycome na MALDI-TOF, ahubwo binatanga amakuru mashya kubyerekeye ibishobora kuvamo karubone ya hydrata muri biomass ya lignocellulosique.
Twasesenguye kandi isukari yibigize agace ka oligosaccharide dukoresheje isukari ya TMS.Twifashishije GC-MS, twahisemo ibice bigize isukari ya neural (idakomoka) hamwe nisukari ya aside (GluA na GalA) mubice bya oligosaccharide (Ishusho 5).Acide Glucuronic iboneka mu bice bya acide C na D, naho aside galacturonic iboneka mu bice bya aside A na B, byombi bikaba ari DP nyinshi bigize isukari ya aside.Ibisubizo ntabwo byemeza gusa amakuru yacu ya ELISA na MALDI, ariko kandi birahuye nubushakashatsi bwatubanjirije bwo kwegeranya oligosaccharide.Kubwibyo, twizera ko uburyo bwa immunologiya bugezweho dukoresheje biotinylation ya oligosaccharide hamwe no gusuzuma ELISA nyuma birahagije kugirango hamenyekane oligosaccharide ya soluble yongeye kuboneka mubushakashatsi butandukanye bwibinyabuzima.
Kuva ELISA ishingiye kuri mAb uburyo bwo gusuzuma bwemejwe nuburyo butandukanye, twashakaga kurushaho gushakisha ubushobozi bwubu buryo bushya bwo kubara.Ibicuruzwa bibiri bya oligosaccharide, xylohexasaccharide oligosaccharide (XHE) na 23-α-L-arabinofuranosyl-xylotriose (A2XX), byaguzwe kandi bipimishwa hakoreshejwe uburyo bushya bwa mAb bwibasiye urukuta rw'akagari glycan.Igishushanyo cya 6 cyerekana isano iri hagati yikimenyetso cya biotinylated ihuza ibimenyetso hamwe nubunini bwibiti bya oligosaccharide, byerekana uburyo bwa adsorption ya Langmuir.Muri mAbs, CCRC-M137, CCRC-M138, CCRC-M147, CCRC-M148, na CCRC-M151 bifitanye isano na XHE, na CCRC-M108, CCRC-M109, na LM11 bifitanye isano na A2XX hejuru ya 1 nm kugeza 100 nano.Bitewe no kuboneka kwa antibodi mugihe cyibigeragezo, ubushakashatsi buke bwakozwe hamwe na buri oligosaccharide yibanze.Aha twakwibutsa ko antibodies zimwe na zimwe zifata muburyo butandukanye kuri oligosaccharide imwe na substrate, byashoboka ko ihuza epitopa zitandukanye kandi zishobora kuba zifitanye isano zitandukanye.Uburyo hamwe nibisobanuro byerekana neza epitope bizamenyekana cyane mugihe uburyo bushya bwa mAb bwakoreshejwe mubitegererezo nyabyo.
Ibicuruzwa bibiri bya oligosaccharide byakoreshejwe kugirango hamenyekane urwego rwo kumenya mAbs zitandukanye.Hano, umurongo uhuza hamwe na log yibanze ya oligosaccharide yerekana uburyo bwa Langmuir adsorption ya (A) XHE hamwe na mAb na (B) A2XX hamwe na mAb.Epitopes ijyanye nayo yerekana imiterere ya oligosaccharide yubucuruzi ikoreshwa nka substrate muri assay.
Gukoresha antibodiyite ya glycan yibasiwe na glycocomic (isesengura rya glycocomic cyangwa ELISA ishingiye kuri mAb isuzuma) nigikoresho gikomeye cyo kuranga byimbitse kuranga igice kinini cyurukuta runini rwa glycans rugizwe na biomass yibimera.Nyamara, isesengura rya glycan risanzwe riranga urukuta runini rwa glycans, kubera ko oligosaccharide nyinshi zidahagarikwa neza ku byapa bya ELISA.Muri ubu bushakashatsi, ububiko bwibigori bwateguwe na AFEX bwashizwemo hydrolyz mu buryo bukomeye.Isesengura ryisukari ryakoreshejwe kugirango hamenyekane ibice bigize urukuta rwa karubone nziza ya hydrolyzate.Nyamara, isesengura rya mAb isesengura rya oligosakisaride ntoya muri hydrolysates ntirisuzugurwa, kandi harakenewe ibikoresho byongeweho kugirango uhagarike neza oligosakisaride ku byapa bya ELISA.
Turabamenyesha hano uburyo bushya kandi bunoze bwa oligosaccharide immobilisation yo gusuzuma mAb muguhuza biotinylation ya oligosaccharide ikurikirwa na ELISA kwerekanwa kuri plaque ya NeutrAvidin.Biotinylated oligosaccharide yerekana ubudahangarwa bwerekanaga isano ihagije ya antibody kugirango ishobore gutahura vuba kandi neza oligosaccharide.Isesengura ryibigize ziriya oligosaccharide zinangiye zishingiye kuri mass spectrometrie yemeje ibyavuye muri ubu buryo bushya bwo gukingira indwara.Niyo mpamvu, ubu bushakashatsi bwerekana ko guhuza biotinylation ya oligosaccharide na ELISA hamwe na antibodiyite ya monoclonal yibasiwe na glycan bishobora gukoreshwa kugirango hamenyekane aho bihurira na oligosaccharide kandi birashobora gukoreshwa cyane mubundi bushakashatsi bwibinyabuzima buranga imiterere ya oligosaccharide.
Ubu buryo bushingiye kuri biotine bushingiye kuri glycan ni bwo raporo ya mbere ishoboye gukora iperereza ku karuboni ya karubone ya hydratitant ya oligosaccharide ikemuka muri biomass y'ibimera.Ibi bifasha kumva impamvu ibice bimwe na bimwe bya biomass binangiye cyane kubijyanye no kubyara ibinyabuzima.Ubu buryo bwuzuza icyuho cyingenzi muburyo bwo gusesengura glycome kandi bugura uburyo bukoreshwa muburyo bwagutse bwa substrate irenze ibimera oligosaccharide.Mu bihe biri imbere, dushobora gukoresha robotike ya biotinylation kandi tugakoresha uburyo twateje imbere kugirango dusesengure byinshi byisesengura dukoresheje ELISA.
Ibyatsi by'ibigori (CS) byakuwe mu mbuto za Hybrid Pioneer 33A14 byasaruwe mu 2010 bivuye mu murima wa Kramer i Ray, muri Kolorado.Uruhushya rwa nyir'ubutaka, iyi biomass irashobora gukoreshwa mubushakashatsi. Ingero zabitswe zumye <6% ubuhehere mu mifuka ya zip-gufunga ubushyuhe bwicyumba. Ingero zabitswe zumye <6% ubuhehere mu mifuka ya zip-gufunga ubushyuhe bwicyumba. Об କାନцы хранились сух мин при влажности <6% в пакакх с застежкой-молнией при комнатной мае. Ingero zabitswe zumye kuri <6% yubushuhe mumifuka ya zipper mubushyuhe bwicyumba.6 在 室温 下 以 干燥 <6% 的 水分 储存 在 自封袋 中。6 在 室温 下 以 干燥 <6% Об କାନцы хранят в пак сех застежкой-молнией при комнатной мае с влажностью <6%. Ingero zibikwa mumifuka ya zipper mubushyuhe bwicyumba hamwe nubushuhe <6%.Ubushakashatsi bwubahirije amabwiriza y’ibanze n’igihugu.Isesengura ryakozwe ryakozwe hakoreshejwe protocole ya NREL.Ibigize wasangaga birimo glucan 31.4%, 18.7% xylan, 3,3% arabinan, 1,2% galactan, 2,2% acetyl, 14.3% lignine, proteine ​​1,7% na 13. 4% ivu.
Cellic® CTec2 (138 mg protein / ml, ubufindo VCNI 0001) ni uruvange rugoye rwa selile, β-glucosidase na Cellic® HTec2 (157 mg protein / ml, lot VHN00001) kuva Novozymes (Franklinton, NC, USA)).Multifect Pectinase® (72 mg protein / mL), uruvange runini rwimisemburo ya pectine itesha agaciro, yatanzwe na DuPont Industrial Biosciences (Palo Alto, CA, USA).Intungamubiri za poroteyine za Enzyme zagenwe no kugereranya ibirimo poroteyine (no gukuramo umusanzu wa azote itari poroteyine) ukoresheje isesengura rya azote ya Kjeldahl (uburyo bwa AOAC 2001.11, Dairy One Cooperative Inc, Ithaca, NY, USA).Isi ya Diatomaceous 545 yaguzwe muri EMD Millipore (Billerica, MA).Carbone ikora (DARCO, 100 mesh granules), Avicel (PH-101), beech xylan, nindi miti yose yaguzwe muri Sigma-Aldrich (St. Louis, MO).
Kwiyitirira AFEX byakorewe muri GLBRC (Laboratoire y'Ubushakashatsi ya Biomass, MSU, Lansing, MI, USA).Mbere yo kuvura byakorewe kuri 140 ° C. mu minota 15.46 igihe cyo gutura ku kigereranyo cya 1: 1 cya ammoniya ya anhidrous na biomass kuri 60% (w / w) yapakiye mumashanyarazi yicyuma kitagira ingese (Isosiyete ya Parr Instruments Company).Byatwaye iminota 30.Imashini yazanywe kuri 140 ° C hanyuma ammonia irekurwa byihuse, bituma biomass isubira vuba mubushyuhe bwicyumba.Ibigize AFEX yabanje gutunganyirizwa mu bigori (ACS) byari bisa n’ibyavuwe ibigori bitavuwe (UT-CS).
Ibikomeye cyane ACSH 25% (w / w) (hafi 8% yipakurura dextran) byateguwe nkibikoresho byo gutangiza umusaruro munini wa oligosakisaride.Enzymatique hydrolysis ya ACS yakozwe hifashishijwe imvange yubucuruzi yubucuruzi irimo Cellic® Ctec2 10 mg protein / g glucan (muri biomass yabigenewe), Htec2 (Novozymes, Franklinton, NC), 5 mg protein / g glucan, na Multifect Pectinase (Genencor Inc, USA).).), 5 mg protein / g dextran.Hydrolysis ya Enzymatique yakorewe muri bioreactor ya litiro 5 ifite ubunini bwa litiro 3, pH 4.8, 50 ° C na 250 rpm.Nyuma ya hydrolysis mumasaha 96, hydrolyzate yakusanyirijwe hamwe na centrifugation kuri 6000 rpm kuminota 30 hanyuma saa 14000 rpm kuminota 30 kugirango ikureho ibidafite ingufu.Hydrolyzate yahise ikorerwa filteri ya sterile ikoresheje akayunguruzo ka mm 0.22.Hydrolizate iyungurujwe yabitswe mu macupa ya sterile kuri 4 ° C. hanyuma igabanywa kuri karubone.
Isesengura ryibigize ibimera bishingiye ku binyabuzima bikurikije uburyo bwo gusesengura laboratoire ya NREL: gutegura ingero zo gusesengura ibihimbano (NREL / TP-510-42620) no kumenya karubone ya hydrata na lignine muri biomass (NREL / TP-510 - 42618) 47.
Isesengura rya Oligosaccharide yumugezi wa hydrolyzate ryakozwe ku gipimo cya ml 2 hakoreshejwe uburyo bwa hydrolysis bushingiye kuri autoclave.Kuvanga urugero rwa hydrolyzate hamwe na 69.7 µl ya 72% ya acide sulfurike muri ml 10 ya screw cap cap culture hanyuma ukabika kuri 1 h ku ntebe ya 121 ° C, ukonjesha ku rubura hanyuma ukayungurura mumashanyarazi akomeye ya chromatografiya (HPLC).Ubwinshi bwa oligosaccharide bwagenwe no gukuramo ubunini bwa monosaccharide mu cyitegererezo kitari hydrolyzed uhereye ku isukari yuzuye mu cyitegererezo cya aside-hydrolyzed.
Glucose, xylose, na arabinose yibanze muri acide hydrolysed biomass yasesenguwe hifashishijwe sisitemu ya Shimadzu HPLC ifite ibikoresho bya autosampler, icyuma gishyushya inkingi, pompe ya gitewokarasi, hamwe na detektori yerekana ibintu ku nkingi ya Bio-Rad Aminex HPX-87H.Inkingi yagumishijwe kuri 50 ° C hanyuma isukurwa 0,6 ml / min 5 mM H2SO4 mumazi.gutemba.
Indengakamere ya hydrolyzate yahinduwe kandi isesengurwa kubintu bya monomer na oligosaccharide.Isukari ya Monomeric yabonetse nyuma ya hydrolysis ya enzymatique yasesenguwe na HPLC ifite ibikoresho bya Bio-Rad (Hercules, CA) Aminex HPX-87P n'inkingi yo kurinda ivu.Ubushyuhe bwinkingi bwagumishijwe kuri 80 ° C, amazi yakoreshejwe nkicyiciro kigendanwa gifite umuvuduko wa 0,6 ml / min.Oligosaccharide yagenwe na hydrolysis muri acide ya dilute kuri 121 ° C ukurikije uburyo bwasobanuwe muri ref.41, 48, 49.
Isesengura rya Sakaride ryakozwe ku mbuto mbisi, AFEX yabanje kuvurwa hamwe n’ibisigazwa bya biomass byose bidafite hydrolysed (harimo no gukora ibivamo urukuta rw'utugingo ngengabuzima no gusuzuma mAb) hakoreshejwe uburyo bwasobanuwe mbere 27, 43, 50, 51.Kugira ngo isesengura rya glycome, ibisigisigi bidashobora gushonga by'ibikoresho by'urukuta rw'ibimera byateguwe bivuye mu bisigazwa bya biomass kandi bigakurwa mu buryo bwa serie hamwe na reagent zigenda ziyongera nka ammonium oxalate (50 mM), karubone ya sodium (50 mM na 0.5% w / v), CON.(1M na 4M, byombi hamwe na 1% w / v sodium borohydride) na chlorite ya aside nkuko byasobanuwe 52,53.Ibikururwa byaje gukorerwa ELISA kurwanya akantu katoroshye ka mAb50s kerekejwe ku rukuta rw'akagari glycan, hanyuma reaction ya mAb yerekanwe nk'ikarita y'ubushyuhe.mAbs yibasira urukuta rw'utugingo ngengabuzima glycan yaguzwe mu bubiko bwa laboratoire (CCRC, JIM na MAC).
Intambwe imwe biotinylation ya oligosaccharide.Guhuza karubone hamwe na biotine-LC-hydrazide byakozwe hakoreshejwe uburyo bukurikira.Biotin-LC-hydrazide (4,6 mg / 12 μ mol) yashongeshejwe muri dimethyl sulfoxide (DMSO, 70 μl) no gukurura cyane no gushyushya kuri 65 ° C. mu minota 1.Acide acetike ya glacial (30 µl) yongewemo hanyuma imvange isukwa kuri sodium cyanoborohydride (6.4 mg / 100 µmol) hanyuma irashonga rwose nyuma yo gushyuha kuri 65 ° C. muminota 1.Noneho, kuva 5 kugeza 8 μl zivanze na reaction zongewe kuri oligosaccharide yumye (1-100 nmol) kugirango ubone inshuro 10 cyangwa nyinshi zirenze urugero za label hejuru yo kugabanya impera.Igisubizo cyakorewe kuri 65 ° C kuri 2 h, nyuma yicyitegererezo gihita cyezwa.Nta sodium cyanoborohydride yakoreshejwe mubushakashatsi bwanditseho nta kugabanya, kandi ingero zakozwe kuri 65 ° C. mumasaha 2.5.
ELISA gutwika no gukaraba ingero za biotinylated oligosaccharide.25 μl z'icyitegererezo cya biotinylated (100 μl ya buri cyitegererezo cyegeranijwe kivanze muri ml 5 ya 0.1 M ya Tris buffer igisubizo (TBS)) yongewe kuri buri riba rya plaque yashizwemo avidin.Amariba yo kugenzura yashizwemo 50 μl ya biotine ku gipimo cya 10 μg / ml muri 0.1 M TBS.Amazi ya Deionised yakoreshejwe nk'igifuniko cyo gupima ubusa.Ikibaho cyashyizwemo amasaha 2 mubushyuhe bwicyumba mu mwijima.Koza isahani inshuro 3 hamwe na 0.1% byamata asukuye muri 0.1 M TBS ukoresheje gahunda no.11 kuri Grenier igorofa 3A.
Kwiyongera no gukaraba antibodi yibanze.Ongeramo 40 µl ya antibody yibanze kuri buri riba.Shyiramo microplate kumasaha 1 mubushyuhe bwicyumba mwijimye.Isahani yogejwe inshuro 3 n'amata 0.1% muri 0.1M TBS ukoresheje gahunda yo gukaraba # 11 kuri Grenier Flat 3A.
Ongeramo antibody ya kabiri hanyuma ukarabe.Ongeramo 50 µl yimbeba / imbeba ya kabiri (ivanze 1: 5000 mumata 0.1% muri 0.1 M TBS) kuri buri riba.Shyiramo microplate kumasaha 1 mubushyuhe bwicyumba mwijimye.Microplates noneho yogejwe inshuro 5 n'amata 0.1% muri 0.1 M TBS ukoresheje gahunda yo gukaraba isahani ya Grenier Flat 5A.
Ongeraho substrate.Ongeramo 50 µl ya 3,3 ′, 5,5′-tetramethylbenzidine (TMB) kuri substrate y'ibanze (wongeyeho ibitonyanga 2 bya buffer, ibitonyanga 3 bya TMB, ibitonyanga 2 bya hydrogen peroxide kuri ml 15 y'amazi ya deioni).Tegura substrate ya TMB.na vortex mbere yo gukoresha).Shyiramo microplate mubushyuhe bwicyumba muminota 30.Mu mwijima.
Uzuza intambwe hanyuma usome ikibaho.Ongeramo 50 µl ya 1 N acide sulfurike kuri buri riba hanyuma wandike iyinjira kuva 450 kugeza 655 nm ukoresheje umusomyi wa ELISA.
Tegura ibisubizo 1 mg / ml byiyi analyite mumazi ya deionised: arabinose, rhamnose, fucose, xylose, aside galacturonic (GalA), aside glucuronic (GlcA), mannose, glucose, galactose, lactose, N-acetylmannosamine (manNAc), N-acetylglucosamine.(glcNAc), N-acetylgalactosamine (galNAc), inositol (igipimo cyimbere).Ibipimo bibiri byateguwe hongerwaho ibisubizo 1 mg / mL ibisubizo byisukari bigaragara mu mbonerahamwe ya 1. Ingero zarahagaritswe kandi zitwa lyofilize kuri -80 ° C. kugeza amazi yose akuweho (mubisanzwe amasaha agera kuri 12-18).
Ongeramo 100-500 µg by'icyitegererezo kuri screw cap tubes kuringaniza yisesengura.Andika amafaranga wongeyeho.Nibyiza gushonga icyitegererezo muburyo bwihariye bwa solvent hanyuma ukayongeramo umuyoboro nka aliquot y'amazi.Koresha 20 µl ya 1 mg / ml inositol nkibipimo byimbere kuri buri cyitegererezo.Ingano yimbere yimbere yongeweho icyitegererezo igomba kuba imwe nubunini bwimbere bwongewe kumuyoboro usanzwe.
Ongeramo ml 8 ya methanol ya anhydrous kuri flake ya screw.Noneho ml 4 ya 3 N. methanolike HCl igisubizo, ifashwe kandi iranyeganyega.Iyi nzira ntabwo ikoresha amazi.
Ongeramo 500 µl ya 1 M HCl methanol igisubizo kuri oligosaccharide ntangarugero hamwe na TMS isanzwe.Ingero zashizwemo ijoro ryose (amasaha 168) kuri 80 ° C. mumashanyarazi.Kama ibicuruzwa bya methanolysis mubushyuhe bwicyumba ukoresheje ibyuma byumye.Ongeramo 200 µl MeOH hanyuma wongere wumuke.Iyi nzira isubirwamo kabiri.Ongeramo 200 µl ya methanol, 100 µl ya pyridine na 100 µl ya anhydride ya acetike kuri sample hanyuma uvange neza.Ingero zashyizwe mubushyuhe bwicyumba muminota 30.akuma.Ongeramo 200 µl ya methanol hanyuma wongere wumuke.
Ongeramo 200 µl ya Tri-Sil hamwe nubushyuhe bwafashwe muminota 20.80 ° C, hanyuma akonje kugeza ubushyuhe bwicyumba.Koresha ibyuma byumye kugirango urusheho gukama icyitegererezo kugeza kuri 50 µl.Ni ngombwa kumenya ko tutemereye ko ingero zuma burundu.
Ongeramo ml 2 ya hexane hanyuma uvange neza na vortexing.Uzuza inama za pipete ya Pasteur (5-8 mm) hamwe nigice cyubwoya bwikirahure winjizamo ubwoya bwikirahure hejuru ya pipeti ya diametre 5-3 / 4.Ingero zashyizwe kuri 3000 g muminota 2.Ibisigisigi byose bidashobora gushonga biragwa.Kama icyitegererezo kuri 100-150 µl.Ingano igera kuri 1 μl yatewe muri GC-MS ku bushyuhe bwa mbere bwa 80 ° C nigihe cyambere cyiminota 2.0 (Imbonerahamwe 2).