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Chotchinga magazi-ubongo ndi chotchinga magazi-ubongo zimalepheretsa mankhwala ochiritsira matenda a ubongo kuti asafike pa zolinga zawo m'thupi la mitsempha, motero zimalepheretsa chithandizo chabwino cha matenda amitsempha. Kuti tipeze zinthu zatsopano zonyamula ubongo m'thupi, tinayambitsa laibulale ya T7 phage peptide ndi magazi ndi madzi a m'mitsempha (CSF) zomwe zinasonkhanitsidwa motsatizana pogwiritsa ntchito chitsanzo cha makoswe akuluakulu odziwa bwino ntchito. Ma clones enieni a phage adachulukitsidwa kwambiri mu CSF pambuyo posankha maulendo anayi. Kuyesa kwa ma peptides apadera kunawonetsa kuchuluka kwa CSF kopitilira 1000. Kugwira ntchito kwa peptide-mediated delivery ku ubongo kunatsimikiziridwa ndi kuchepa kwa 40% kwa amyloid-β mumadzimadzi a m'mitsempha pogwiritsa ntchito BACE1 peptide inhibitor yolumikizidwa ndi peptide yatsopano yodziwika. Zotsatirazi zikusonyeza kuti ma peptides omwe adadziwika ndi njira zosankhidwira phage m'thupi akhoza kukhala njira zothandiza popereka ma macromolecules ku ubongo ndi mphamvu yochiritsira.
Kafukufuku wokhudza chithandizo cha CNS wayang'ana kwambiri pakupeza mankhwala ndi zinthu zomwe zimagwira ntchito bwino zomwe zimasonyeza mphamvu zowongolera CNS, komanso khama lochepa pakupeza njira zomwe zimathandizira kutumiza mankhwala ku ubongo. Izi zikuyamba kusintha tsopano chifukwa kutumiza mankhwala, makamaka mamolekyu akuluakulu, ndi gawo lofunika kwambiri pakukula kwa mankhwala a neuroscience amakono. Malo a dongosolo la mitsempha lapakati amatetezedwa bwino ndi dongosolo lotchinga mitsempha yamagazi, lomwe limapangidwa ndi chotchinga cha magazi-ubongo (BBB) ​​​​ndi chotchinga cha magazi-ubongo (BCBB)1, zomwe zimapangitsa kuti zikhale zovuta kupereka mankhwala ku ubongo1,2. Akuti pafupifupi mankhwala onse akuluakulu a mamolekyu ndi mankhwala opitilira 98% ang'onoang'ono a mamolekyu amachotsedwa mu ubongo3. Ichi ndichifukwa chake ndikofunikira kwambiri kuzindikira njira zatsopano zoyendera ubongo zomwe zimapereka chithandizo chabwino komanso chodziwikiratu cha mankhwala ku CNS 4,5. Komabe, BBB ndi BCSFB zimaperekanso mwayi wabwino kwambiri woperekera mankhwala pamene akulowa ndikulowa m'mapangidwe onse a ubongo kudzera mu mitsempha yake yayikulu. Motero, khama lomwe likuchitika panopa logwiritsa ntchito njira zosagwiritsa ntchito mankhwala osokoneza bongo ku ubongo limadalira kwambiri njira yotumizira mauthenga (PMT) pogwiritsa ntchito cholandirira cha BBB6 chochokera ku endogenous. Ngakhale kuti posachedwapa pakhala kupita patsogolo kwakukulu pogwiritsa ntchito njira ya transferrin receptor7,8, pakufunika kupititsa patsogolo njira zatsopano zotumizira mauthenga zomwe zili ndi makhalidwe abwino. Pachifukwa ichi, cholinga chathu chinali kupeza ma peptide omwe angathe kuyendetsa CSF transport, chifukwa angagwiritsidwe ntchito popereka ma macromolecules ku CNS kapena kutsegula njira zatsopano zolandirira mauthenga. Makamaka, ma receptor ndi ma transporter a cerebrovascular system (BBB ndi BSCFB) amatha kukhala ngati zolinga zomwe zingatheke popereka mankhwala othandiza komanso ochiritsira. Madzi a m'mitsempha ya ubongo (CSF) ndi chinthu chobisika cha choroid plexus (CS) ndipo chimakhudzana mwachindunji ndi madzi a m'mitsempha ya ubongo kudzera mu subarachnoid space ndi ventricular space4. Posachedwapa zawonetsedwa kuti madzi a m'mitsempha ya ubongo ya subarachnoid amafalikira kwambiri mu interstitium ya ubongo9. Tikukhulupirira kuti tidzalowa mu malo a parenchymal pogwiritsa ntchito njira yolowera ya subarachnoid iyi kapena kudzera mwachindunji kudzera mu BBB. Kuti tikwaniritse izi, takhazikitsa njira yolimba yosankhira phage mu vivo yomwe imadziwika bwino ma peptides omwe amayendetsedwa ndi imodzi mwa njira ziwirizi zosiyana.
Tsopano tikufotokoza njira yotsatizana yowunikira ma phage mu vivo pogwiritsa ntchito CSF ​​sampling pamodzi ndi high throughput sequencing (HTS) kuti tiwonetsetse magawo oyamba osankhidwa ndi mitundu yosiyanasiyana ya laibulale. Kuwunika kunachitika pa makoswe odziwa bwino ntchito pogwiritsa ntchito canula yayikulu ya cisterna (CM) yokhazikika kuti tipewe kuipitsidwa kwa magazi. Chofunika kwambiri, njira iyi imasankha brain-target ndi ma peptides omwe ali ndi ntchito yoyendera kudutsa chotchinga cha mitsempha yamagazi. Tinagwiritsa ntchito ma T7 phages chifukwa cha kukula kwawo kochepa (~60 nm)10 ndipo tinanena kuti ndi oyenera kunyamula ma vesicles omwe amalola kudutsa kwa endothelial ndi/kapena epithelial-medulla chotchinga. Pambuyo pa maulendo anayi a panning, ma phages adapezeka kuti akuwonetsa kuti CSF ​​in vivo ndi mgwirizano wamphamvu wa mitsempha yamagazi. Chofunika kwambiri, tinatha kutsimikizira zomwe tapeza powonetsa kuti ma peptides abwino kwambiri omwe amasankhidwa komanso opangidwa ndi mankhwala amatha kunyamula katundu wa mapuloteni kupita ku madzi a m'magazi. Choyamba, zotsatira za mankhwala a CNS zinakhazikika mwa kuphatikiza peptide yotsogola yotumizira ndi choletsa cha peptide ya BACE1. Kuwonjezera pa kusonyeza kuti njira zowunikira ntchito mu vivo zimatha kuzindikira ma peptide atsopano otumizira ubongo ngati zonyamulira zonyamula mapuloteni ogwira ntchito, tikuyembekeza kuti njira zofanana zosankhira ntchito nazonso zikhale zofunika pozindikira njira zatsopano zotumizira ubongo.
Kutengera ndi mayunitsi opangira ma plaque (PFU), pambuyo pa gawo la kulongedza ma phage, laibulale ya ma peptide a T7 phage a 12-mer omwe ali ndi mitundu yosiyanasiyana ya pafupifupi 109 idapangidwa ndikupangidwa (onani Zipangizo ndi Njira). Ndikofunikira kudziwa kuti tidasanthula mosamala laibulale iyi tisanagwiritse ntchito ma primer osinthidwa. Kukulitsa kwa PCR kwa zitsanzo za laibulale ya phage pogwiritsa ntchito ma primer osinthidwa kunapanga ma amplicons omwe anali ogwirizana mwachindunji ndi HTS (Chithunzi Chowonjezera 1a). Chifukwa cha a) zolakwika za HTS11 sequencing, b) kukhudza mtundu wa ma primer (NNK)1-12, ndi c) kupezeka kwa wild-type (wt) phage (skeleton inserts) mu laibulale yoyimirira, njira yosefera sequence idakhazikitsidwa kuti ichotse chidziwitso chotsimikizika cha sequence (Chithunzi Chowonjezera 1b). Njira zosefera izi zimagwira ntchito ku malaibulale onse a HTS sequencing. Pa laibulale yokhazikika, ziwerengero zokwana 233,868 zinapezeka, zomwe 39% mwa izo zinadutsa muyezo wa zosefera ndipo zinagwiritsidwa ntchito pofufuza ndi kusankha laibulale pazotsatira (Chithunzi Chowonjezera 1c–e). Zomwe zinawerengedwazo zinali zochulukitsa za ma base pairs atatu kutalika ndi nsonga ya ma nucleotide 36 (Chithunzi Chowonjezera 1c), kutsimikizira kapangidwe ka laibulale (NNK) 1-12. Chodziwika bwino n'chakuti, pafupifupi 11% ya mamembala a laibulale anali ndi PAGISRELVDKL backbone ya 12-dimensional wild-type (wt) backbone, ndipo pafupifupi theka la magawo (49%) anali ndi zoikamo kapena zochotsedwa. HTS ya laibulale ya laibulale inatsimikizira kusiyanasiyana kwakukulu kwa ma peptide mu laibulale: oposa 81% a magawo a peptide adapezeka kamodzi kokha ndipo 1.5% yokha idapezeka m'makopi ≥4 (Chithunzi Chowonjezera 2a). Mafupipafupi a amino acid (aa) m'malo onse 12 mu repertoire adagwirizana bwino ndi mafupipafupi omwe amayembekezeredwa pa chiwerengero cha ma codon opangidwa ndi repertoire ya NKK yofooka (Chithunzi Chowonjezera 2b). Mafupipafupi omwe adawonedwa a aa residues omwe adalembedwa ndi izi adagwirizana bwino ndi mafupipafupi owerengedwa (r = 0.893) (Chithunzi Chowonjezera 2c). Kukonzekera malaibulale a phage kuti alowetsedwe kumaphatikizapo njira zokulitsa ndi kuchotsa endotoxin. Izi zawonetsedwa kale kuti zitha kuchepetsa kusiyanasiyana kwa malaibulale a phage12,13. Chifukwa chake, tidakonza laibulale ya phage yowonjezeredwa ndi mbale yomwe idachotsedwa endotoxin ndikuyiyerekeza ndi laibulale yoyambirira kuti tiyerekeze kuchuluka kwa AA. Kugwirizana kwamphamvu (r = 0.995) kudawonedwa pakati pa dziwe loyambirira ndi dziwe lowonjezeredwa ndi loyeretsedwa (Chithunzi Chowonjezera 2d), zomwe zikusonyeza kuti mpikisano pakati pa ma clones omwe amakulitsidwa pama mbale pogwiritsa ntchito phage ya T7 sunachititse tsankho lalikulu. Kuyerekeza kumeneku kumachokera pa kuchuluka kwa ma motif a tripeptide mu laibulale iliyonse, popeza kusiyanasiyana kwa malaibulale (~109) sikungathe kujambulidwa mokwanira ngakhale ndi HTS. Kusanthula pafupipafupi kwa aa pamalo aliwonse kunawonetsa kusagwirizana pang'ono komwe kumadalira malo m'malo atatu omaliza a repertoire yomwe yalowetsedwa (Chithunzi Chowonjezera 2e). Pomaliza, tinatsimikiza kuti mtundu ndi kusiyanasiyana kwa laibulale zinali zovomerezeka ndipo kusintha kochepa kokha kwa kusiyanasiyana kunawonedwa chifukwa cha kukulitsa ndi kukonzekera malaibulale a phage pakati pa maulendo angapo osankhidwa.
Kusankha madzi a m'mitsempha ya m'mitsempha ya m'mitsempha kungachitike mwa kuika cannula mu CM ya makoswe ozindikira kuti azitha kuzindikira T7 phage yomwe yalowetsedwa m'mitsempha (iv) kudzera mu BBB ndi/kapena BCSFB (Chithunzi 1a-b). Tinagwiritsa ntchito manja awiri odziyimira pawokha (manja A ndi B) m'magawo atatu oyamba a kusankha mu vivo (Chithunzi 1c). Pang'onopang'ono tinawonjezera mphamvu ya kusankha mwa kuchepetsa kuchuluka kwa phage komwe kwayambitsidwa m'magawo atatu oyamba a kusankha. Pa gawo lachinayi la panning, tinaphatikiza zitsanzo kuchokera ku nthambi A ndi B ndikuchita zisankho zitatu zina zodziyimira pawokha. Kuti tiphunzire za mawonekedwe a tinthu ta T7 phage mu chitsanzo ichi, wild-type phage (PAGISRELVDKL master insert) idalowetsedwa m'makoswe kudzera m'mitsempha ya mchira. Kubwezeretsa ma phage kuchokera ku madzi a m'mitsempha ya m'mitsempha ndi magazi nthawi zosiyanasiyana kunawonetsa kuti ma phage ang'onoang'ono a T7 icosahedral anali ndi gawo loyambirira lotseguka mwachangu kuchokera m'magazi (Chithunzi Chowonjezera 3). Kutengera ndi ma titers omwe adaperekedwa ndi kuchuluka kwa magazi a makoswe, tinawerengera kuti pafupifupi 1% yokha ya wt. phage kuchokera ku mlingo womwe adaperekedwa idapezeka m'magazi mphindi 10 pambuyo pa jakisoni wa m'mitsempha. Pambuyo pa kuchepa koyambirira kumeneku, kuchotsedwa pang'onopang'ono kwa primary kunayesedwa ndi theka la moyo wa mphindi 27.7. Chofunika kwambiri, ma phages ochepa okha ndi omwe adatengedwa kuchokera ku CSF compartment, zomwe zikusonyeza kuti palibe maziko okwanira oti ma phages amtundu wachilengedwe alowe mu CSF compartment (Chithunzi Chowonjezera 3). Pa avareji, pafupifupi 1 x 10-3% ya ma titers a T7 phage m'magazi ndi 4 x 10-8% ya ma phages omwe adalowetsedwa poyamba adapezeka mu cerebrospinal fluid panthawi yonse yoyeserera (0-250 min). Chodziwika bwino ndichakuti, theka la moyo (25.7 min) la wild-type phage mu cerebrospinal fluid linali lofanana ndi lomwe limapezeka m'magazi. Deta iyi ikuwonetsa kuti chotchinga chomwe chimalekanitsa gawo la CSF ndi magazi chilipo m'makoswe omwe ali ndi CM-cannulated, zomwe zimathandiza kuti ma laibulale a phage asankhidwe m'thupi kuti adziwe ma clones omwe amasamutsidwa mosavuta kuchokera m'magazi kupita ku gawo la CSF.
(a) Kukhazikitsa njira yopezeranso cerebrospinal fluid (CSF) kuchokera ku dziwe lalikulu. (b) Chithunzi chosonyeza malo a selo la chotchinga cha mitsempha yapakati (CNS) ndi njira yosankhidwira yomwe imagwiritsidwa ntchito kuzindikira ma peptide omwe amadutsa chotchinga cha magazi-ubongo (BBB) ​​​​ndi chotchinga cha magazi-ubongo. (c) Tchati chowunikira kayendedwe ka phage mu vivo. Mu gawo lililonse losankhidwa, ma phage (zozindikiritsa nyama mkati mwa mivi) adabayidwa kudzera m'mitsempha. Nthambi ziwiri zodziyimira pawokha (A, B) zimasungidwa padera mpaka gawo lachinayi losankhidwa. Pa magawo osankhidwa 3 ndi 4, phage clone iliyonse yochotsedwa ku CSF idasankhidwa pamanja. (d) Kinetics ya phage yochotsedwa m'magazi (mabwalo ofiira) ndi cerebrospinal fluid (ma triangles obiriwira) panthawi yoyamba yosankhidwa m'makoswe awiri otsekedwa pambuyo pobayidwa m'mitsempha ya laibulale ya T7 peptide (ma phage awiri/nyama 1012). Mabwalo abuluu amasonyeza kuchuluka koyambirira kwa phage m'magazi, kowerengedwa kuchokera ku kuchuluka kwa phage yolowetsedwa, poganizira kuchuluka kwa magazi onse. Mabwalo akuda amasonyeza malo olumikizirana a mzere wa y wotengedwa kuchokera ku kuchuluka kwa phage m'magazi. (e,f) Onetsani kuchuluka ndi kufalikira kwa ma motif onse a tripeptide omwe akupezeka mu peptide. Chiwerengero cha ma motif omwe apezeka mu mawerengedwe 1000 chawonetsedwa. Chofunika kwambiri (p < 0.001) ma motif olemeretsedwa ali ndi madontho ofiira. (e) Kufananiza kwa scatterplot kuyerekeza kuchuluka kwa scatterplot ya tripeptide ya laibulale yolowetsedwa ndi phage yochokera m'magazi kuchokera ku nyama #1.1 ndi #1.2. (f) Kufananiza kwa scatterplot kuyerekeza kuchuluka kwa scatterplot ya nyama #1.1 ndi #1.2 yopezeka m'magazi ndi madzi am'mitsempha. (g, h) Kuyimira kwa Sequence ID ya phage yolemeretsedwa m'magazi (g) motsutsana ndi malaibulale olowetsedwa ndi phage yolemeretsedwa mu CSF (h) motsutsana ndi magazi pambuyo pa kusankha kwa in vivo mwa nyama zonse ziwiri. Kukula kwa code ya chilembo chimodzi kumasonyeza kuti amino acid imeneyo imapezeka kangati pamalo amenewo. Chobiriwira = polar, purple = neutral, buluu = basic, wofiira = acidic ndi wakuda = hydrophobic amino acids. Chithunzi 1a, b chinapangidwa ndi kupangidwa ndi Eduard Urich.
Tinalowetsa laibulale ya phage peptide m'makoswe awiri a CM instrument (clades A ndi B) ndi phage yolekanitsidwa kuchokera ku cerebrospinal fluid ndi magazi (Chithunzi 1d). Kutuluka koyambirira mwachangu kwa laibulale kunali kochepa poyerekeza ndi phage yamtundu wa wild-type. Hafu ya moyo wa laibulale yolowetsedwa m'zinyama zonse ziwiri inali mphindi 24.8 m'magazi, mofanana ndi phage yamtundu wa wild-type, ndi mphindi 38.5 mu CSF. Zitsanzo za phage zamagazi ndi cerebrospinal fluid kuchokera ku nyama iliyonse zinayesedwa HTS ndipo ma peptide onse odziwika adawunikidwa kuti awone ngati pali tripeptide motif yayifupi. Ma Tripeptide motifs adasankhidwa chifukwa amapereka maziko ochepa opangira kapangidwe ndi kuyanjana kwa peptide-protein14,15. Tapeza mgwirizano wabwino pakugawidwa kwa ma motifs pakati pa laibulale ya phage yolowetsedwa ndi ma clones otengedwa m'magazi a nyama zonse ziwiri (Chithunzi 1e). Deta ikuwonetsa kuti kapangidwe ka laibulale kamangoleredwa pang'ono m'magazi. Ma frequency a amino acid ndi ma consensus sequences adawunikidwanso pamalo aliwonse pogwiritsa ntchito kusintha kwa pulogalamu ya Weblogo16. Chochititsa chidwi n'chakuti, tinapeza kuchuluka kwamphamvu kwa glycine m'magazi (Chithunzi 1g). Pamene magazi ankayerekezeredwa ndi ma clone osankhidwa kuchokera ku CSF, kusankha kwamphamvu ndi kuchotsedwa kwa ma motifs kunawonedwa (Chithunzi 1f), ndipo ma amino acid ena analipo makamaka pamalo omwe adakonzedweratu mu 12-member (Chithunzi 1h). Chodziwika bwino n'chakuti, nyama iliyonse inali yosiyana kwambiri mu cerebrospinal fluid, pomwe glycine m'magazi inawonedwa mu nyama zonse ziwiri (Chithunzi Chowonjezera 4a-j). Pambuyo pofufuza mosamala deta ya sequence mu cerebrospinal fluid ya nyama #1.1 ndi #1.2, ma peptides 964 ndi 420 apadera a 12-mer adapezeka (Chithunzi Chowonjezera 1d-e). Ma clone a phage odzipatula adakulitsidwa ndikupatsidwa gawo lachiwiri la kusankha kwa in vivo. Phage yotengedwa kuchokera ku gawo lachiwiri la kusankha idayikidwa mu HTS mu nyama iliyonse ndipo ma peptide onse odziwika adagwiritsidwa ntchito ngati cholowera mu pulogalamu yozindikira motif kuti iwunike momwe ma motif a tripeptide amachitikira (Chithunzi 2a, b, ef). Poyerekeza ndi kuzungulira koyamba kwa phage yomwe idachotsedwa ku CSF, tidawona kusankha kwina ndi kuchotsa kusankha kwa ma motif ambiri mu CSF m'nthambi A ndi B (Chithunzi 2). Njira yodziwira netiweki idagwiritsidwa ntchito kuti idziwe ngati akuyimira mitundu yosiyanasiyana ya mndandanda wogwirizana. Kufanana komveka bwino kudawonedwa pakati pa ma sequence a 12-dimensional omwe adabwezedwa ndi CSF mu alternative clade A (Chithunzi 2c, d) ndi clade B (Chithunzi 2g, h). Kusanthula kwa grouped mu nthambi iliyonse kudawonetsa ma profiles osiyanasiyana osankhidwa a ma peptide 12-mer (Chithunzi Chowonjezera 5c, d) ndi kuwonjezeka kwa CSF/blood titer ratio pakapita nthawi kwa ma clones ophatikizidwa pambuyo pa kuzungulira kwachiwiri kwa kusankha poyerekeza ndi kuzungulira koyamba kwa kusankha (Chithunzi Chowonjezera 5e).).
Kuwonjezeka kwa ma motif ndi ma peptide mu cerebrospinal fluid ndi maulendo awiri otsatizana a kusankha kwa in vivo functional phage display.
Ma phage onse a cerebrospinal fluid omwe adapezeka kuchokera ku gawo loyamba la nyama iliyonse (nyama #1.1 ndi #1.2) adayikidwa pamodzi, kukulitsidwa, kutsatizana kwa HT ndikulowetsedwanso pamodzi (2 x 1010 phages/nyama) makoswe awiri a SM omwe adalowetsedwa mu cannulation (#1.1 → #). 2.1 ndi 2.2, 1.2 → 2.3 ndi 2.4). (a,b,e,f) Ma scatterplot ogwirizana omwe akuyerekeza kuchuluka kwa ma motif a tripeptide a ma phages onse ochokera ku CSF mu gawo loyamba ndi lachiwiri losankha. Kuchuluka kwa ma motif ndi kufalikira kwa ma motif omwe akuyimira ma tripeptide onse omwe angapezeke mu ma peptides m'njira zonse ziwiri. Chiwerengero cha ma motif omwe apezeka mu ma readings 1000 akuwonetsedwa. Ma motif omwe adasankhidwa kwambiri (p < 0.001) kapena osaphatikizidwa mu imodzi mwa malaibulale ofananizidwa awonetsedwa ndi madontho ofiira. (c, d, g, h) Chizindikiro cha sequence cha ma amino acid 12 okhala ndi CSF okhala ndi ma amino acid 12 ataliatali kutengera ma round 2 ndi 1 a in vivo select. Kukula kwa code ya chilembo chimodzi kumasonyeza kuti amino acid imeneyo imapezeka kangati pamalo amenewo. Kuti iimirire logo, kuchuluka kwa ma CSF sequences otengedwa kuchokera ku nyama iliyonse pakati pa ma round awiri osankhidwa kumayerekezeredwa ndipo ma round olemera mu round yachiwiri akuwonetsedwa: (c) #1.1–#2.1 (d) #1.1–#2.2 (g) #1.2–#2.3 ndi (h) #1.2–#2.4. Ma amino acid olemera kwambiri pamalo operekedwa mu (c, d) nyama nambala 2.1 ndi nambala 2.2 kapena (g, h) mu nyama nambala 2.3 ndi nambala 2.4 akuwonetsedwa mu mtundu. Chobiriwira = polar, purple = neutral, blue = basic, red = acidic ndi black = hydrophobic amino acids.
Pambuyo pa gawo lachitatu la kusankha, tinapeza ma peptide sequences 124 apadera (#3.1 ndi #3.2) ochokera ku ma clone 332 a CSF-reconstituted phage olekanitsidwa ndi nyama ziwiri (Chithunzi Chowonjezera 6a). Ma LGSVS (18.7%) anali ndi chiwerengero chapamwamba kwambiri, kutsatiridwa ndi ma wild-type inserts PAGISRELVDKL (8.2%), MRWFFSHASQGR (3%), DVAKVS (3%), TWLFSLG (2.2%), ndi SARGSWREIVSLS (2.2%). Mu gawo lomaliza lachinayi, tinasonkhanitsa nthambi ziwiri zosankhidwa paokha kuchokera ku nyama zitatu zosiyana (Chithunzi 1c). Mwa ma clone 925 a phage sequenced omwe adapezeka kuchokera ku CSF, mu gawo lachinayi tinapeza ma peptide sequences 64 apadera (Chithunzi Chowonjezera 6b), pakati pawo chiwerengero cha wild-type phage chinatsika kufika pa 0.8%. Ma clones odziwika bwino a CSF mu gawo lachinayi anali LYVLHSRGLWGFKLAAALE (18%), LGSVS (17%), GFVRFRLSNTR (14%), KVAWRVFSLFWK (7%), SVHGV (5%), GRPQKINGARVC (3.6%) ndi RLSSVDSDLSGC (3, 2%). %). Kutalika kwa ma peptides osankhidwa kumachitika chifukwa cha kulowetsedwa/kuchotsedwa kwa nucleotide kapena ma codons oima msanga mu ma primer a laibulale pogwiritsa ntchito ma codons owonongeka pakupanga laibulale ya NNK. Ma codons oima msanga amapanga ma peptides afupiafupi ndipo amasankhidwa chifukwa ali ndi aa motif yabwino. Ma peptides ataliatali angachitike chifukwa cha kulowetsedwa/kuchotsedwa mu ma primer a malaibulale opangidwa. Izi zimayika stop codon yopangidwa kunja kwa chimango ndikuiwerenga mpaka stop codon yatsopano iwonekere pansi. Kawirikawiri, tinawerengera zinthu zowonjezera pamitundu yonse inayi yosankhidwa poyerekeza deta yolowera ndi deta yotulutsa zitsanzo. Pa nthawi yoyamba yowunikira, tinagwiritsa ntchito ma wild-type phage titers ngati chizindikiro chosadziwika bwino. Chochititsa chidwi n'chakuti, kusankha kwa phage kolakwika kunali kolimba kwambiri mu nthawi yoyamba ya CSF, koma osati m'magazi (Chithunzi 3a), zomwe zitha kukhala chifukwa cha mwayi wochepa wa kufalikira kwa ziwalo zambiri za peptide laibulale mu CSF compartment kapena relative phages nthawi zambiri zimasungidwa bwino kapena kuchotsedwa m'magazi kuposa bacteriophages. Komabe, mu nthawi yachiwiri yowunikira, kusankha kwamphamvu kwa phages mu CSF kunawonedwa m'magulu onse awiri, zomwe zikusonyeza kuti nthawi yapitayi inali yolemera mu phages zomwe zikuwonetsa ma peptide omwe amalimbikitsa kutengedwa kwa CSF (Chithunzi 3a). Apanso, popanda kuchuluka kwa magazi. Komanso mu nthawi yachitatu ndi yachinayi, ma phage clones anali olemera kwambiri mu CSF. Poyerekeza kuchuluka kwa peptide iliyonse yapadera pakati pa nthawi ziwiri zomaliza zosankhidwa, tinapeza kuti nthawiyo inali yolemera kwambiri mu nthawi yachinayi yosankhidwa (Chithunzi 3b). Ma motif 931 a tripeptide adachotsedwa mu ma peptide onse 64 apadera pogwiritsa ntchito njira zonse ziwiri za peptide. Ma motif olemera kwambiri mu gawo lachinayi adawunikidwa bwino kuti adziwe momwe amawonjezerera m'magawo onse poyerekeza ndi laibulale yolowetsedwa (kudula: 10% yowonjezera) (Chithunzi Chowonjezera 6c). Mapangidwe ambiri osankhidwa adawonetsa kuti zolinga zambiri zomwe zidaphunziridwa zidachulukira m'magawo onse am'mbuyomu a nthambi zonse ziwiri zosankhidwa. Komabe, ma motif ena (monga SGL, VSG, LGS GSV) anali ochokera ku alternative clade A, pomwe ena (monga FGW, RTN, WGF, NTR) adachulukira mu alternative clade B.
Kutsimikizira kwa kayendedwe ka CSF kwa ma peptide owonetsedwa ndi CSF olemera ndi ma peptide otsogola a biotinylated omwe amalumikizidwa ku streptavidin payloads.
(a) Ma ratio a Enrichment omwe amawerengedwa m'magawo onse anayi (R1-R4) kutengera ma titer a jekeseni (input = I) phage (PFU) ndi ma titer a CSF phage omwe adatsimikizika (output = O). Zinthu zowonjezera m'magawo atatu omaliza (R2-R4) zidawerengedwa poyerekeza ndi kuzungulira kwapitako ndi kuzungulira koyamba (R1) ndi deta ya kulemera. Mipiringidzo yotseguka ndi madzi am'mitsempha, mipiringidzo yokhala ndi mithunzi ndi plasma. (***p<0.001, kutengera mayeso a t-test a Wophunzira). (b) Mndandanda wa ma peptide a phage ochulukirapo, osankhidwa malinga ndi kuchuluka kwawo poyerekeza ndi ma phage onse omwe asonkhanitsidwa mu CSF pambuyo pa kuzungulira 4 kwa kusankha. Ma clones asanu ndi limodzi odziwika bwino a phage amawonetsedwa mumitundu, manambala ndi zinthu zowonjezera pakati pa kuzungulira 3 ndi 4 kwa kusankha (insets). (c,d) Ma clones asanu ndi limodzi a phage olemera kwambiri, ma laibulale a empty phage ndi ma parent phage peptide ochokera kuzungulira 4 adawunikidwa payekhapayekha mu chitsanzo cha CSF sampling. CSF ndi zitsanzo za magazi zidasonkhanitsidwa panthawi yomwe yasonyezedwa. (c) Kuchuluka kofanana kwa ma clone 6 a phage (2 x 1010 phages/nyama), ma phage opanda kanthu (#1779) (2 x 1010 phages/nyama) ndi malaibulale a peptide a phage (2 x 1012 phages/nyama) Jambulani osachepera 3 CM kwa nyama yodulidwayo padera kudzera m'mitsempha ya mchira. Mankhwala a CSF a laibulale iliyonse ya phage clone ndi phage peptide pakapita nthawi akuwonetsedwa. (d) akuwonetsa chiŵerengero chapakati cha CSF/magazi a ma phage/mL onse obwezeretsedwa panthawi yopereka zitsanzo. (e) Ma peptide anayi otsogola opangidwa ndi synthetic ndi control imodzi yosweka adalumikizidwa ndi biotin ku streptavidin kudzera mu N-terminus yawo (tetramer display) kutsatiridwa ndi jakisoni (mtsempha wa mchira iv, 10 mg streptavidin/kg). Makoswe osachepera atatu oikidwa m'mimba (N = 3). ). Zitsanzo za CSF zinasonkhanitsidwa panthawi yomwe yasonyezedwa ndipo kuchuluka kwa streptavidin kunayesedwa ndi CSF anti-streptavidin ELISA (ndi = sikunapezeke). (*p<0.05, **p<0.01, ***p<0.001, kutengera mayeso a ANOVA). (f) Kuyerekeza kwa amino acid sequence ya most riched phage peptide clone #2002 (wofiirira) ndi ma clones ena osankhidwa a phage peptide kuchokera ku round yachinayi ya kusankha. Zidutswa za amino acid zofanana ndi zofanana zimayikidwa mitundu.
Mwa ma phage onse olemera mu gawo lachinayi (Chithunzi 3b), ma clone asanu ndi limodzi omwe adasankhidwa adasankhidwa kuti apitirize kusanthula payekha mu chitsanzo cha CSF sampling. Kuchuluka kofanana kwa ma phage asanu ndi limodzi omwe adasankhidwa, phage yopanda kanthu (yopanda insert) ndi malaibulale a prophage peptide adalowetsedwa m'zinyama zitatu za CM zosungidwa m'ma cannulated, ndipo pharmacokinetics idapezeka mu CSF (Chithunzi 3c) ndi magazi (Chithunzi Chowonjezera 7). Ma phage clones onse omwe adayesedwa adayang'ana gawo la CSF pamlingo wokwera nthawi 10-1000 kuposa wa empty control phage (#1779). Mwachitsanzo, ma clones #2020 ndi #2077 anali ndi ma CSF titers okwana pafupifupi 1000 kuposa control phage. Mbiri ya pharmacokinetic ya peptide iliyonse yosankhidwa ndi yosiyana, koma onse ali ndi CSF homing ability yayikulu. Tinaona kuchepa kosalekeza pakapita nthawi kwa ma clones #1903 ndi #2011, pomwe kwa ma clones #2077, #2002 ndi #2009 kuwonjezeka mkati mwa mphindi 10 zoyambirira kungasonyeze kuti munthu akuyenda bwino koma kuyenera kutsimikiziridwa. Ma Clones #2020, #2002, ndi #2077 adakhazikika pamlingo wapamwamba, pomwe kuchuluka kwa CSF kwa ma clone #2009 kunachepa pang'onopang'ono pambuyo pa kuwonjezeka koyamba. Kenako tinayerekeza kuchuluka kwa CSF iliyonse ndi kuchuluka kwake m'magazi (Chithunzi 3d). Kugwirizana kwa mean titer ya CSF iliyonse ndi titer yake yamagazi nthawi zonse zoyesedwa kunawonetsa kuti atatu mwa ma species asanu ndi limodzi anali ndi CSF yambiri m'magazi. Chosangalatsa ndichakuti, clone #2077 inawonetsa kukhazikika kwa magazi (Chithunzi Chowonjezera 7). Kuti titsimikizire kuti ma peptide okha amatha kunyamula katundu wina kupatula tinthu ta phage kupita ku CSF compartment, tinapanga ma peptide anayi otsogola omwe adachokera ku biotin pa N-terminus komwe ma peptide amalumikizana ndi tinthu ta phage. Ma peptides a Biotinylated (nos. 2002, 2009, 2020 ndi 2077) adalumikizidwa ndi streptavidin (SA) kuti apeze mitundu yosiyanasiyana yofanana ndi geometry ya phage. Mtundu uwu unatithandizanso kuyeza kufalikira kwa SA m'magazi ndi madzi am'mitsempha ngati ma peptides a mapuloteni onyamula katundu. Chofunika kwambiri, deta ya phage nthawi zambiri imatha kubwerezedwanso pamene ma peptide opangidwa adaperekedwa mu mtundu uwu wa SA-conjugated (Chithunzi 3e). Ma peptides osweka anali ndi kufalikira kochepa koyamba komanso CSF ​​yofulumira yokhala ndi milingo yosawoneka mkati mwa maola 48. Kuti tidziwe bwino njira zoperekera ma peptides a phage awa mu malo a CSF, tinasanthula komwe ma peptide a phage amagunda pogwiritsa ntchito immunohistochemistry (IHC) amachitikira kuti azindikire mwachindunji tinthu ta phage ola limodzi titalandira jekeseni m'mitsempha. Chodziwika bwino n'chakuti, ma clones #2002, #2077, ndi #2009 amatha kupezeka ndi utoto wamphamvu m'mitsempha yamagazi ya ubongo, pomwe control phage (#1779) ndi clone #2020 sizinapezeke (Chithunzi Chowonjezera 8). Izi zikusonyeza kuti ma peptide awa amathandizira ku ubongo mwachindunji podutsa BBB. Kusanthula kwatsatanetsatane kumafunika kuti ayesere lingaliro ili, chifukwa njira ya BSCFB ingakhalenso yokhudzidwa. Poyerekeza mndandanda wa amino acid wa clone wolemera kwambiri (#2002) ndi ma peptide ena osankhidwa, zidadziwika kuti ena mwa iwo ali ndi ma amino acid ofanana, omwe angasonyeze njira yofananira yoyendera (Chithunzi 3f).
Chifukwa cha mawonekedwe ake apadera a plasma komanso kuwonjezeka kwakukulu kwa CSF pakapita nthawi, phage display clone #2077 idafufuzidwanso kwa nthawi yayitali ya maola 48 ndipo idatha kubwerezanso kuwonjezeka kwachangu kwa CSF komwe kumawoneka mogwirizana ndi milingo yokhazikika ya SA (Chithunzi 4a). Ponena za ma phage clones ena omwe adadziwika, #2077 idapaka utoto kwambiri pamitsempha yamagazi yaubongo ndipo idawonetsa colocalization yayikulu ndi capillary marker lectin ikawonedwa bwino kwambiri komanso mwina utoto wina mu parenchymal space (Chithunzi 4b). Kuti tifufuze ngati zotsatira za peptide-mediated pharmacological zitha kupezeka mu CNS, tidachita kuyesera komwe mitundu ya biotinylated ya i) transit peptide #2077 ndi ii) BACE1 inhibitor peptide idasakanizidwa ndi SA pamitundu iwiri yosiyana. Pa kuphatikiza kumodzi tidagwiritsa ntchito BACE1 peptide inhibitor yokha ndipo kwa inayo tidagwiritsa ntchito chiŵerengero cha 1:3 cha BACE1 peptide inhibitor ndi #2077 peptide. Zitsanzo zonse ziwiri zinaperekedwa kudzera m'mitsempha ndipo kuchuluka kwa beta-amyloid peptide 40 (Abeta40) m'magazi ndi m'mitsempha ya ubongo kunayesedwa pakapita nthawi. Abeta40 inayesedwa mu CSF chifukwa imasonyeza kuletsa kwa BACE1 mu ubongo parenchyma. Monga momwe zinalili, ma complex onsewa anachepetsa kwambiri kuchuluka kwa Abeta40 m'magazi (Chithunzi 4c, d). Komabe, zitsanzo zokha zomwe zili ndi chisakanizo cha peptide nambala 2077 ndi choletsa peptide ya BACE1 yolumikizidwa ndi SA zinapangitsa kuti Abeta40 ichepe kwambiri mu madzi a m'mitsempha ya ubongo (Chithunzi 4c). Deta ikuwonetsa kuti peptide nambala 2077 imatha kunyamula puloteni ya 60 kDa SA kupita ku CNS komanso imayambitsa zotsatira za mankhwala pogwiritsa ntchito zoletsa za SA-conjugated za peptide ya BACE1.
(a) Kujambulira kwa clonal (2 × 10 phages/nyama) kwa T7 phage kusonyeza ma profiles a pharmacokinetic a CSF peptide #2077 (RLSSVDSDLSGC) ndi uninjected control phage (#1779) mwa makoswe osachepera atatu okhala ndi CM. (b) Chithunzi cha confocal microscopic cha mitsempha yaying'ono ya cortical yoyimira m'makoswe olowetsedwa ndi phage (2 × 10 10 phages/nyama) kusonyeza counterdating ya peptide #2077 ndi mitsempha yamagazi (lectin). Ma clones a phage awa adaperekedwa kwa makoswe atatu ndipo adaloledwa kuzungulira kwa ola limodzi asanalowetsedwe. Ubongo unadulidwa ndikupakidwa utoto ndi ma antibodies a polyclonal FITC motsutsana ndi T7 phage capsid. Mphindi khumi musanalowetsedwe ndi kukhazikika pambuyo pake, DyLight594-labeled lectin idaperekedwa kudzera m'mitsempha. Zithunzi zowala zomwe zikuwonetsa utoto wa lectin (wofiira) wa mbali ya kuwala kwa mitsempha yaying'ono ndi ma phage (obiriwira) mu lumen ya mitsempha yamagazi ndi minofu ya ubongo ya perivascular. Chipilalacho chikufanana ndi 10 µm. (c, d) Peptide yoletsa BACE1 ya Biotinylated yokha kapena yophatikizidwa ndi biotinylated transit peptide #2077 inalumikizidwa ndi streptavidin kutsatiridwa ndi jakisoni wa m'mitsempha wa makoswe osachepera atatu a CM otsekedwa (10 mg streptavidin/kg). Kuchepetsa kwa BACE1 peptide inhibitor-mediated mu Aβ40 kunayesedwa ndi Aβ1-40 ELISA m'magazi (ofiira) ndi madzi a m'mitsempha (lalanje) panthawi yomwe yasonyezedwa. Kuti zimveke bwino, mzere wokhala ndi madontho umajambulidwa pa graph pa sikelo ya 100%. (c) Kuchepa kwa Aβ40 m'magazi (ma triangle ofiira) ndi madzi a m'mitsempha ya ...
Kuwonetsa phage kwagwiritsidwa ntchito bwino m'magawo angapo a kafukufuku wa zamankhwala17. Njirayi yagwiritsidwa ntchito pa maphunziro osiyanasiyana a mitsempha ya m'thupi18,19 komanso maphunziro okhudza mitsempha ya m'magazi20,21,22,23,24,25,26. Mu kafukufukuyu, tawonjezera kugwiritsa ntchito njira iyi yosankhira osati kokha kuzindikira mwachindunji ma peptide omwe amayang'ana mitsempha ya m'magazi, komanso kupeza anthu omwe ali ndi katundu woyendetsa wodutsa chotchinga cha magazi ndi ubongo. Tsopano tikufotokoza za chitukuko cha njira yosankhira mu thupi la m'thupi mwa makoswe okhala ndi CM ndikuwonetsa kuthekera kwake kuzindikira ma peptide omwe ali ndi CSF homing properties. Pogwiritsa ntchito T7 phage yomwe ikuwonetsa laibulale ya ma peptides 12-mer random, tinatha kuwonetsa kuti T7 phage ndi yaying'ono mokwanira (pafupifupi 60 nm m'mimba mwake)10 kuti igwirizane ndi chotchinga cha magazi ndi ubongo, motero kudutsa mwachindunji chotchinga cha magazi ndi ubongo kapena choroid plexus. Tinaona kuti kusonkhanitsa CSF kuchokera ku makoswe a CM omwe ali ndi cannula kunali njira yowunikira bwino yomwe imayendetsedwa bwino mu vivo, ndipo phage yotulutsidwayo sinali yongolumikizana ndi mitsempha yamagazi komanso inkagwira ntchito ngati chonyamulira kudutsa chotchinga cha magazi ndi ubongo. Kuphatikiza apo, posonkhanitsa magazi nthawi imodzi ndikugwiritsa ntchito HTS ku CSF ​​ndi ma phage otengedwa m'magazi, tinatsimikizira kuti kusankha kwathu CSF sikunakhudzidwe ndi kuchuluka kwa magazi kapena kuyenerera kwa kukula pakati pa maulendo osankhidwa. Komabe, gawo la magazi ndi gawo la njira yosankhira, popeza ma phage omwe amatha kufika ku gawo la CSF ayenera kukhala ndi moyo ndikuzungulira m'magazi nthawi yayitali kuti adzichulukitse muubongo. Kuti tipeze zambiri zodalirika kuchokera ku data ya HTS yaiwisi, tinakhazikitsa zosefera zomwe zidasinthidwa ku zolakwika za sequencing za pulatifomu mu ntchito yosanthula. Mwa kuphatikiza magawo a kinetic mu njira yowunikira, tinatsimikizira pharmacokinetics yachangu ya ma phage amtundu wa T7 (t½ ~ 28 min) m'magazi24, 27, 28 komanso tinazindikira theka la moyo wawo mu cerebrospinal fluid (t½ ~ 26 min) pamphindi). Ngakhale kuti pali ma pharmacokinetics ofanana m'magazi ndi CSF, 0.001% yokha ya kuchuluka kwa phage m'magazi ndi yomwe ingapezeke mu CSF, zomwe zikusonyeza kuyenda kochepa kwa phage yamtundu wa wild-type T7 kudutsa chotchinga cha magazi ndi ubongo. Ntchitoyi ikuwonetsa kufunika kwa kusankha koyamba pogwiritsa ntchito njira zoyezera magazi m'thupi, makamaka machitidwe a phage omwe amachotsedwa mwachangu kuchokera ku kayendedwe ka magazi, chifukwa ma clone ochepa amatha kufika ku CNS compartment. Chifukwa chake, mu round yoyamba, kuchepa kwa kusiyanasiyana kwa laibulale kunali kwakukulu kwambiri, chifukwa ma clone ochepa okha ndi omwe adasonkhanitsidwa mu chitsanzo chokhwima kwambiri cha CSF. Njira iyi yoyezera magazi m'thupi idaphatikizapo njira zingapo zosankhira monga kusonkhanitsa mwachangu mu compartment ya CSF, kupulumuka kwa ma clone m'magazi, ndikuchotsa mwachangu ma clone a T7 phage m'magazi mkati mwa mphindi 10 zoyambirira (Chithunzi 1d ndi Chithunzi Chowonjezera 4M). ). Chifukwa chake, pambuyo pa round yoyamba, ma clone osiyanasiyana a phage adapezeka mu CSF, ngakhale dziwe loyambirira lomwelo lidagwiritsidwa ntchito pa nyama iliyonse. Izi zikusonyeza kuti njira zingapo zosankhira mosamala malaibulale oyambira omwe ali ndi mamembala ambiri a laibulale zimapangitsa kuti kusiyana kuchepe kwambiri. Chifukwa chake, zochitika zosayembekezereka zidzakhala gawo lofunikira kwambiri pakusankha koyamba, zomwe zimakhudza kwambiri zotsatira zake. N'zotheka kuti ma clones ambiri mu laibulale yoyambirira anali ndi chizolowezi chofanana kwambiri cha CSF. Komabe, ngakhale pansi pa mikhalidwe yoyesera yomweyi, zotsatira za kusankha zitha kusiyana chifukwa cha kuchuluka kochepa kwa ma clones aliwonse mu gulu loyambirira.
Ma motifs olemera mu CSF amasiyana ndi omwe ali m'magazi. Chosangalatsa n'chakuti, tawona kusintha koyamba kupita ku ma peptides olemera mu glycine m'magazi a nyama iliyonse. (Chithunzi 1g, Zithunzi Zowonjezera 4e, 4f). Ma peptides okhala ndi phage okhala ndi glycine akhoza kukhala okhazikika komanso osatheka kuchotsedwa m'magazi. Komabe, ma peptides olemera mu glycine awa sanapezeke m'zitsanzo zamadzimadzi a m'mitsempha ya ubongo, zomwe zikusonyeza kuti malaibulale osankhidwa adadutsa magawo awiri osiyana osankhidwa: limodzi m'magazi ndi lina lololedwa kusonkhana mumadzimadzi a m'mitsempha ya ubongo. Ma clones olemera mu CSF omwe adachokera ku gawo lachinayi la kusankha adayesedwa kwambiri. Pafupifupi ma clones onse oyesedwa payekha adatsimikiziridwa kuti ali ndi CSF poyerekeza ndi blank control phage. Kugunda kwa peptide imodzi (#2077) kudawunikidwa mwatsatanetsatane. Idawonetsa theka la moyo wautali poyerekeza ndi ma hits ena (Chithunzi 3d ndi Chithunzi Chowonjezera 7), ndipo chosangalatsa n'chakuti, peptide iyi inali ndi cysteine ​​​​residue ku C-terminus. Posachedwapa zawonetsedwa kuti kuwonjezera kwa cysteine ​​​​ku ma peptides kungathandize kukonza mphamvu zawo za pharmacokinetic mwa kumangirira ku albumin 29. Izi sizikudziwika pakadali pano za peptide #2077 ndipo zimafuna kuphunzira kwina. Ma peptide ena adawonetsa kudalira kwa valence mu CSF enrichment (deta siinawonetsedwe), zomwe zitha kukhala zokhudzana ndi mawonekedwe a pamwamba a T7 capsid. Dongosolo la T7 lomwe tidagwiritsa ntchito lidawonetsa makope 5-15 a peptide iliyonse pa tinthu ta phage. IHC idachitidwa pa ma candidate lead phage clones omwe adalowetsedwa m'mitsempha mu cerebral cortex ya makoswe (Chithunzi Chowonjezera 8). Deta idawonetsa kuti ma clones osachepera atatu (Nambala 2002, Nambala 2009 ndi Nambala 2077) adalumikizana ndi BBB. Pakadali pano kuti tidziwe ngati kuyanjana kwa BBB kumeneku kumabweretsa kusonkhanitsa kwa CSF kapena kuyenda kwa ma clones awa mwachindunji ku BCSFB. Chofunika kwambiri, tikuwonetsa kuti ma peptide osankhidwa amasunga mphamvu yawo yoyendera ya CSF akapangidwa ndikumangiriridwa ku protein cargo. Kumangirira ma peptide a N-terminal biotinylated ku SA kwenikweni kumabwereza zotsatira zomwe zapezeka ndi ma clones awo a phage m'magazi ndi madzi a m'mitsempha ya ubongo (Chithunzi 3e). Pomaliza, tikuwonetsa kuti lead peptide #2077 imatha kulimbikitsa ntchito ya ubongo ya biotinylated peptide inhibitor ya BACE1 yolumikizidwa ku SA, zomwe zimapangitsa kuti pakhale zotsatira zoyipa za pharmacodynamic mu CNS mwa kuchepetsa kwambiri milingo ya Abeta40 mu CSF (Chithunzi 4). Sitinathe kuzindikira ma homologues aliwonse mu database pochita kafukufuku wa peptide sequence homology wa ma hits onse. Ndikofunikira kudziwa kuti kukula kwa laibulale ya T7 ndi pafupifupi 109, pomwe kukula kwa laibulale ya theoretical kwa 12-mers ndi 4 x 1015. Chifukwa chake, tinasankha kachigawo kakang'ono kokha ka malo osiyanasiyana a laibulale ya 12-mer peptide, zomwe zingatanthauze kuti ma peptides okonzedwa bwino amatha kuzindikirika poyesa malo oyandikana nawo a ma hits awa odziwika. Mwachiyelezgero, chimodzi mwa zifukwa zomwe sitinapeze ma homologues achilengedwe a ma peptide awa ndi kusasankhidwa panthawi ya kusintha kwa zinthu kuti tipewe kulowa kosalamulirika kwa ma peptide ena muubongo.
Poganizira zonsezi, zotsatira zathu zimapereka maziko a ntchito yamtsogolo kuti tizindikire ndikufotokozera njira zoyendera za chotchinga cha mitsempha ya ubongo mu vivo mwatsatanetsatane. Kukhazikitsa koyambira kwa njira iyi kumadalira njira yosankhira yomwe sikuti imangozindikira ma clones omwe ali ndi mphamvu zomangira mitsempha ya ubongo, komanso ikuphatikiza gawo lofunika kwambiri lomwe ma clones opambana ali ndi ntchito yolowera kuti adutse zopinga zamoyo mu vivo kupita ku CNS compartment. Ndiko kufotokoza momwe ma peptides awa amayendera komanso zomwe amakonda polumikizana ndi microvasculature yeniyeni kudera la ubongo. Izi zitha kutsogolera kupeza njira zatsopano zoyendera BBB ndi ma receptor. Tikuyembekeza kuti ma peptides omwe adziwika amatha kumangirira mwachindunji ku ma receptor a mitsempha ya ubongo kapena ku ma ligands ozungulira omwe amatumizidwa kudzera mu BBB kapena BCSFB. Ma peptide vectors omwe ali ndi ntchito yoyendera ya CSF omwe apezeka mu ntchitoyi adzafufuzidwa kwambiri. Pakadali pano tikufufuza za ubongo wa ma peptides awa kuti adziwe momwe amatha kuwolokera BBB ndi/kapena BCSFB. Ma peptide atsopanowa adzakhala zida zamtengo wapatali kwambiri popezera ma receptor kapena njira zatsopano komanso popanga nsanja zatsopano zogwira mtima kwambiri zoperekera ma macromolecules, monga biologics, ku ubongo.
Kuthira madzi mu cisterna yayikulu (CM) pogwiritsa ntchito njira yosinthira yomwe yafotokozedwa kale. Makoswe a Wistar ogonekedwa ndi mankhwala oletsa ululu (200-350 g) anaikidwa pa chipangizo choyeretsera khungu ndipo kudula kwapakati kunapangidwa pamwamba pa mutu wometedwa komanso wokonzedwa bwino kuti chigaza chiwonekere. Bowolani mabowo awiri m'dera la sash yapamwamba ndikumangirira zomangira m'mabowo. Bowo lina linabowoledwa mu lateral occipital crest kuti liwongoleredwe ndi chingwe chosapanga dzimbiri mu CM. Ikani simenti ya mano mozungulira chingwecho ndikuchimangirira ndi zomangira. Pambuyo pojambula ndi kulimbitsa simenti, bala la khungu linatsekedwa ndi suture ya 4/0 supramid. Kuyika bwino kwa chingwecho kumatsimikiziridwa ndi kutuluka kwa madzi a m'mitsempha (CSF) mwadzidzidzi. Chotsani khosweyo pa chipangizo choyeretsera khungu, landirani chisamaliro choyenera pambuyo pa opaleshoni ndikuwongolera ululu, ndikulola kuti achire kwa sabata imodzi mpaka zizindikiro za magazi zitawoneka mu madzi a m'mitsempha. Makoswe a Wistar (Crl:WI/Han) adapezeka ku Charles River (France). Makoswe onse anasungidwa m'malo enaake opanda tizilombo toyambitsa matenda. Kuyesa konse kwa nyama kunavomerezedwa ndi Ofesi ya Zanyama ya Mzinda wa Basel, Switzerland, ndipo kunachitika motsatira Chilolezo cha Zinyama Nambala 2474 (Kuyesa Kuyendetsa Ubongo Mwachangu Poyesa Magawo a Odwala Odwala mu Cerebrospinal Fluid ndi Ubongo wa Mphaka).
Sungani khoswe mosamala ndi CM cannula m'manja. Chotsani Datura mu cannula ndikusonkhanitsa 10 µl ya madzi a m'mitsempha omwe amatuluka okha. Popeza kuti mphamvu ya cannula inachepa, zitsanzo zamadzimadzi a m'mitsempha zomwe sizinawonetse kuipitsidwa kwa magazi kapena kusintha kwa mtundu ndi zomwe zinaphatikizidwa mu kafukufukuyu. Pamodzi, pafupifupi 10-20 μl ya magazi idatengedwa kuchokera ku kudula pang'ono kumapeto kwa mchira kupita ku machubu okhala ndi heparin (Sigma-Aldrich). CSF ndi magazi zinasonkhanitsidwa nthawi zosiyanasiyana pambuyo pobayidwa ndi T7 phage m'mitsempha. Pafupifupi 5-10 μl yamadzimadzi idatayidwa chitsanzo chilichonse cha CSF chisanasonkhanitsidwe, chomwe chikugwirizana ndi kuchuluka kwakufa kwa catheter.
Malaibulale adapangidwa pogwiritsa ntchito vekitala ya T7Select 10-3b monga momwe tafotokozera mu buku la T7Select system (Novagen, Rosenberg et al., InNovations 6, 1-6, 1996). Mwachidule, DNA insert ya 12-mer yosasinthika idapangidwa motere:
Kodoni ya NNK idagwiritsidwa ntchito popewa ma codon awiri oima ndi kuchuluka kwa amino acid mu insert. N ndi chiŵerengero cha equimolar chosakanikirana ndi manja cha nucleotide iliyonse, ndipo K ndi chiŵerengero cha equimolar chosakanikirana ndi manja cha adenine ndi cytosine nucleotides. Madera amodzi omangika adasinthidwa kukhala DNA iwiri yomangika mwa kuwonjezera kulowetsedwa ndi dNTP (Novagen) ndi enzyme ya Klenow (New England Biolabs) mu Klenow buffer (New England Biolabs) kwa maola atatu pa 37°C. Pambuyo pa reaction, DNA iwiri yomangika idabwezedwa ndi EtOH precipitation. DNA yomwe idatuluka idagayidwa ndi ma enzyme oletsa EcoRI ndi HindIII (onse ochokera ku Roche). Insert yomangika ndi yoyeretsedwa (QIAquick, Qiagen) (T4 ligase, New England Biolabs) idalumikizidwa mu chimango kukhala vector ya T7 yomangika kale pambuyo pa amino acid 348 ya jini ya 10B capsid. Zochita za ligation zidasungidwa pa 16°C kwa maola 18 musanayambe kulongedza mu vitro. Kupaka ma phage mu vitro kunachitika motsatira malangizo omwe adaperekedwa ndi T7Select 10-3b cloning kit (Novagen) ndipo yankho la phukusili linakulitsidwa kamodzi kuti lipangidwe pogwiritsa ntchito Escherichia coli (BLT5615, Novagen). Ma lysates adayikidwa mu centrifuge, titrated ndikuzizira pa -80° C. ngati yankho la glycerol.
Kukulitsa kwa PCR mwachindunji kwa zigawo zosinthika za phage zomwe zimakulitsidwa mu msuzi kapena mbale pogwiritsa ntchito ma primer osakanikirana a 454/Roche-amplicon. Choyambira cha fusion cha forward chili ndi ma sequences omwe ali pafupi ndi dera losinthika (NNK) 12 (ma template-specific), GS FLX Titanium Adapter A, ndi ma key sequence a library-base anayi (TCAG) (Chithunzi Chowonjezera 1a):
Choyambira chosinthira cha reverse fusion chilinso ndi biotin yolumikizidwa ku mikanda yojambulira ndipo GS FLX Titanium Adapter B yofunikira pakukulitsa kwa clonal panthawi ya emulsion PCR:
Kenako ma amplicon adayikidwa mu 454/Roche pyrosequencing motsatira 454 GS-FLX Titanium protocol. Pa Sanger sequencing yamanja (Applied Biosystems Hitachi 3730 xl DNA Analyzer), T7 phage DNA idakulitsidwa ndi PCR ndikutsatiridwa ndi ma primer pairs otsatirawa:
Ma insertion ochokera ku ma plaque osiyanasiyana adayikidwa PCR amplification pogwiritsa ntchito Roche Fast Start DNA Polymerase Kit (malinga ndi malangizo a wopanga). Chitani hot start (mphindi 10 pa 95 °C) ndi boost cycles 35 (masekondi 50 pa 95 °C, mphindi 1 pa 50 °C, ndi mphindi 1 pa 72 °C).
Phage kuchokera ku malaibulale, phage wamtundu wa wild-type, phage wopulumutsidwa ku CSF ndi magazi, kapena ma clones amodzi ndi amodzi adakulitsidwa mu Escherichia coli BL5615 mu msuzi wa TB (Sigma Aldrich) kapena m'mbale za 500 cm2 (Thermo Scientific) kwa maola 4 pa 37°C. Phage adachotsedwa m'mbale mwa kutsuka mbale ndi Tris-EDTA buffer (Fluka Analytical) kapena mwa kusonkhanitsa ma plaque ndi nsonga za pipette zoyera. Phage adachotsedwa ku supernatant ya culture kapena extraction buffer ndi round imodzi ya polyethylene glycol (PEG 8000) precipitation (Promega) ndikuyimitsidwanso mu Tris-EDTA buffer.
Phage yowonjezeredwa inachotsedwa kangapo ka 2-3 pogwiritsa ntchito mikanda yochotsera endotoxin (Miltenyi Biotec) asanaperekedwe jekeseni ya m'mitsempha (IV) (500 μl/nyama). Mu gawo loyamba, ma phage 2 × 1012 analowetsedwa; mu gawo lachiwiri, ma phage 2 × 1010; mu gawo lachitatu ndi lachinayi losankhidwa, ma phage 2 × 109 pa nyama iliyonse. Kuchuluka kwa ma phage mu CSF ndi zitsanzo za magazi zomwe zinasonkhanitsidwa panthawi yomwe yasonyezedwa kunatsimikiziridwa ndi kuwerengera kwa plaque malinga ndi malangizo a wopanga (T7Select system manual). Kusankha ma phage kunachitidwa mwa kulowetsa m'mitsempha ya malaibulale oyeretsedwa mumtsempha wam'mbuyo kapena mwa kulowetsanso ma phage otengedwa kuchokera ku CSF kuchokera mu gawo lapitalo losankhidwa, ndipo zokolola zotsatira zinachitidwa pa mphindi 10, mphindi 30, mphindi 60, mphindi 90, mphindi 120, mphindi 180, ndi mphindi 240 motsatana. Kuzungulira konse kwa ma panning a in vivo kunachitika momwe nthambi ziwiri zosankhidwa zinasungidwa padera ndikuwunikidwa pa nthawi ya ma raundi atatu oyamba osankhidwa. Ma phage inserts onse otengedwa kuchokera ku CSF kuchokera ku ma raundi awiri oyamba osankhidwa adachitidwa 454/Roche pyrosequencing, pomwe ma clones onse otengedwa kuchokera ku CSF kuchokera ku ma raundi awiri omaliza osankhidwa adasankhidwa pamanja. Ma phage onse a magazi kuchokera ku raundi yoyamba yosankhidwa adachitidwanso 454/Roche pyrosequencing. Pa kubaya ma phage clones, ma phage osankhidwa adakulitsidwa mu E. coli (BL5615) pa mbale za 500 cm2 pa 37°C kwa maola 4. Ma clones osankhidwa payekhapayekha komanso otsatizana adafalikira mu TB medium. Pambuyo pochotsa ma phage, kuyeretsa ndi kuchotsa endotoxin (monga tafotokozera pamwambapa), ma phage 2 × 1010/nyama mu 300 μl adalowetsedwa m'mitsempha m'mitsempha imodzi yamchira.
Kukonza ndi kusefa deta ya sequence moyenerera. Deta ya 454/Roche yosaphika idasinthidwa kuchoka pa mtundu wa binary standard stream map format (sff) kupita ku mtundu wa Pearson human readeable format (fasta) pogwiritsa ntchito pulogalamu ya vendor. Kukonzanso sequence ya nucleotide kunachitika pogwiritsa ntchito mapulogalamu a C ndi ma script (pulogalamu yosatulutsidwa) monga momwe tafotokozera pansipa. Kusanthula deta yoyamba kumaphatikizapo njira zokhwima zosefera magawo ambiri. Kuti zosefera zowerengedwa zomwe sizinali ndi sequence yovomerezeka ya 12mer insert DNA, zowerengedwazo zidalumikizidwa motsatizana ndi chizindikiro choyambira (GTGATGTCGGGGATCCGAATTCT), chizindikiro choyimitsa (TAAGCTTGCGGCCGCACTCGAGTA) ndi background insert (CCCTGCAGGGATATCCCGGGAGCTCGTCGAC) pogwiritsa ntchito mayeso apadziko lonse a Needleman-Wunsch. alignment imalola mpaka kusagwirizana kawiri pa alignment31. Chifukwa chake, zowerengedwa zopanda ma tag oyambira ndi oima ndi zowerengedwa zomwe zili ndi background inserts, mwachitsanzo, alignments zomwe zimaposa chiwerengero chololedwa cha mismatches, zidachotsedwa mu laibulale. Ponena za ma read otsalawo, N-mer DNA sequence yoyambira chizindikiro choyamba mpaka kumapeto chizindikiro chisanachotsedwe kuchokera ku read sequence yoyambirira ndikukonzedwanso (pano ikutchedwa "insert"). Pambuyo pomasulira insert, gawo pambuyo pa codon yoyamba yoyimitsa kumapeto kwa 5′ ya primer imachotsedwa kuchokera ku insert. Kuphatikiza apo, ma nucleotide omwe amatsogolera ku ma codon osakwanira kumapeto kwa 3′ a primer adachotsedwanso. Kuti muchotse ma insert omwe ali ndi ma background sequences okha, ma insert omasuliridwa omwe amayamba ndi amino acid pattern "PAG" nawonso adachotsedwa. Ma peptides okhala ndi kutalika kwa post-translational kochepera 3 amino acid adachotsedwa ku laibulale. Pomaliza, chotsani redundancy mu insert pool ndikudziwa kuchuluka kwa insert iliyonse yapadera. Zotsatira za kusanthula kumeneku zidaphatikizapo mndandanda wa ma nucleotide sequences (insert) ndi ma frequency awo (read) (Zithunzi Zowonjezera 1c ndi 2).
Kuyika DNA ya Gulu N-mer potengera kufanana kwa sequence: Kuti muchotse zolakwika za 454/Roche-specific sequencing (monga mavuto ndi sequencing homopolymer extensions) ndikuchotsa zochulukirapo zosafunikira kwenikweni, ma sequence a N-mer DNA sequence inserts (inserts) omwe adasefedwa kale amasankhidwa motsatira kufanana. insertions (mpaka maziko awiri osafanana amaloledwa) pogwiritsa ntchito algorithm yobwerezabwereza yomwe imatanthauzidwa motere: ma insertion amasankhidwa kaye ndi ma frequency awo (apamwamba kwambiri mpaka otsika), ndipo ngati ali ofanana, ndi mtundu wawo wachiwiri ndi kutalika (kutalika kwambiri mpaka kufupi) ). Chifukwa chake, ma insertion obwerezabwereza komanso ataliatali kwambiri amatanthauzira "gulu" loyamba. Ma frequency a gulu amayikidwa ku key frequency. Kenako, kuyika kulikonse komwe kwatsala pamndandanda wosankhidwa kunayesedwa kuti kuwonjezedwa ku gululo mwa kuyika Needleman-Wunsch pairwise. Ngati chiwerengero cha mismatch, insertions, kapena deletions mu alignment sichidutsa malire a 2, kuyika kumawonjezedwa ku gululo, ndipo ma frequency onse a gulu amawonjezeka ndi kuchuluka kwa ma insertion omwe awonjezedwa. Ma insertions omwe awonjezedwa ku gulu amalembedwa kuti agwiritsidwa ntchito ndipo amachotsedwa pakukonzedwa kwina. Ngati ndondomeko yolowera siingawonjezedwe ku gulu lomwe lilipo kale, ndondomeko yolowera imagwiritsidwa ntchito popanga gulu latsopano ndi ma frequency oyenera olowera ndipo yalembedwa kuti yagwiritsidwa ntchito. Kubwerezabwereza kumatha pamene ndondomeko iliyonse yolowera yagwiritsidwa ntchito kupanga gulu latsopano kapena ikhoza kuphatikizidwa mu gulu lomwe lilipo kale. Kupatula apo, ma inserts ophatikizidwa omwe ali ndi ma nucleotide pamapeto pake amamasuliridwa kukhala ma peptide sequences (malaibulale a peptide). Zotsatira za kusanthula kumeneku ndi seti ya ma insertion ndi ma frequency awo ofanana omwe amapanga chiwerengero cha ma reads otsatizana (Chithunzi Chowonjezera 2).
Kupanga Motif: Kutengera mndandanda wa ma peptides apadera, laibulale idapangidwa yokhala ndi mitundu yonse ya amino acid (aa) monga momwe zasonyezedwera pansipa. Mtundu uliwonse wotheka wa kutalika kwa 3 unachotsedwa mu peptide ndipo mawonekedwe ake otsutsana adawonjezedwa pamodzi ndi laibulale yodziwika bwino ya motif yokhala ndi mitundu yonse (tripeptides). Malaibulale a mitundu yobwerezabwereza kwambiri adasanjidwa ndipo kuchotsedwa kwa zinthu zina. Kenako, pa tripeptide iliyonse mu laibulale ya motif, tinayang'ana kupezeka kwake mu laibulale pogwiritsa ntchito zida zowerengera. Pankhaniyi, kuchuluka kwa peptide yokhala ndi tripeptide yopezedwa ya motif kumawonjezedwa ndikuperekedwa ku motif mu laibulale ya motif ("chiwerengero cha mitundu"). Zotsatira za kupanga motif ndi gulu la magawo awiri lomwe lili ndi zochitika zonse za tripeptides (motifs) ndi mtengo wawo, zomwe ndi chiwerengero cha kuwerenga kotsatizana komwe kumabweretsa motif yofanana pamene kuwerengako kusefedwa, kugawidwa m'magulu, ndikumasuliridwa. Miyeso monga tafotokozera mwatsatanetsatane pamwambapa.
Kusasintha chiwerengero cha zojambula ndi zithunzi zofanana: Chiwerengero cha zojambula pa chitsanzo chilichonse chinasinthidwa pogwiritsa ntchito
pomwe ni ndi chiwerengero cha kuwerenga komwe kuli mutu i. Chifukwa chake, vi ikuyimira kuchuluka kwa kuwerenga (kapena ma peptide) omwe ali ndi motif i mu chitsanzo. Ma P-values ​​​​a chiwerengero chosakhazikika cha motifs adawerengedwa pogwiritsa ntchito mayeso enieni a Fisher. Ponena za ma correlogram a chiwerengero cha zolinga, ma correlations a Spearman adawerengedwa pogwiritsa ntchito chiwerengero chokhazikika cha zolinga ndi R.
Kuti muwone kuchuluka kwa ma amino acid pamalo aliwonse mu laibulale ya peptide, ma logogram a pa intaneti 32, 33 (http://weblogo.threeplusone.com) adapangidwa. Choyamba, kuchuluka kwa ma amino acid pamalo aliwonse a 12-mer peptide kumasungidwa mu matrix ya 20×12. Kenako, seti ya ma peptide 1000 okhala ndi kuchuluka kwa amino acid komweko pamalo aliwonse amapangidwa mu mtundu wa fasta-sequence ndipo amaperekedwa ngati cholowera ku logo ya 3, yomwe imapanga chithunzi cha kuchuluka kwa ma amino acid pamalo aliwonse. pa laibulale ya peptide yoperekedwa. Kuti muwone ma data amitundu yambiri, mamapu otentha adapangidwa pogwiritsa ntchito chida chopangidwa mkati mwa R (biosHeatmap, phukusi la R lomwe silinatulutsidwe). Ma dendrogram omwe adawonetsedwa m'mapu otentha adawerengedwa pogwiritsa ntchito njira ya Ward yolumikizira magulu ndi metric ya mtunda wa Euclidean. Pa kusanthula ziwerengero za deta yowunikira ma motif, ma P values ​​​​a scoring unnormalized adawerengedwa pogwiritsa ntchito mayeso enieni a Fisher. Ma P-values ​​​​a ma datasets ena adawerengedwa mu R pogwiritsa ntchito mayeso a t-test a Student kapena ANOVA.
Ma phage clones osankhidwa ndi ma phage opanda zoikamo adabayidwa kudzera m'mitsempha kudzera m'mitsempha ya mchira (2 × 1010 phages/nyama mu 300 μl PBS). Mphindi khumi asanaperekedwe magazi ndi kukhazikika pambuyo pake, nyama zomwezo zidabayidwa kudzera m'mitsempha ndi 100 μl ya DyLight594-labeled lectin (Vector Laboratories Inc., DL-1177). Mphindi 60 pambuyo poti aperekedwe magazi, makoswe adabayidwa kudzera mumtima ndi 50 ml PBS kutsatiridwa ndi 50 ml 4% PFA/PBS. Zitsanzo za ubongo zidakonzedwanso usiku wonse mu 4% PFA/PBS ndikunyowa mu 30% sucrose usiku wonse pa 4°C. Zitsanzo zimasungidwa mu chisakanizo cha OCT. Kusanthula kwa immunohistochemical kwa zitsanzo zozizira kunachitika pa kutentha kwa chipinda pa 30 µm cryosections yotsekedwa ndi 1% BSA ndipo inayikidwa ndi ma antibodies a polyclonal FITC-labeled against T7 phage (Novus NB 600-376A) pa 4 °C. Ikani mu incubation usiku wonse. Pomaliza, zigawozo zinatsukidwa katatu ndi PBS ndipo zinafufuzidwa ndi confocal laser microscope (Leica TCS SP5).
Ma peptide onse omwe ali ndi chiyero chocheperako cha 98% adapangidwa ndi GenScript USA, adasinthidwa kukhala biotinylated ndi lyophilized. Biotin imalumikizidwa kudzera mu triple glycine spacer yowonjezera pa N-terminus. Yang'anani ma peptide onse pogwiritsa ntchito mass spectrometry.
Streptavidin (Sigma S0677) inasakanizidwa ndi peptide yothira biotinylated yokwana 5-fold, peptide yothira BACE1 yothira biotinylated, kapena kuphatikiza (3:1 ratio) kwa peptide yothira BACE1 yothira biotinylated ndi peptide yothira BACE1 mu 5–10% DMSO/incubated mu PBS. Ola limodzi kutentha kwa chipinda musanapereke jekeseni. Ma peptide olumikizidwa ndi Streptavidin adabayidwa kudzera m'mitsempha pa mlingo wa 10 mg/kg mu imodzi mwa mitsempha ya mchira ya makoswe omwe ali ndi ubongo.
Kuchuluka kwa ma streptavidin-peptide complexes kunayesedwa ndi ELISA. Ma plates a Nunc Maxisorp microtiter (Sigma) anapakidwa usiku wonse pa 4°C ndi 1.5 μg/ml ya mbewa ya anti-streptavidin antibody (Thermo, MA1-20011). Pambuyo potseka (blocking buffer: 140 nM NaCL, 5 mM EDTA, 0.05% NP40, 0.25% gelatin, 1% BSA) kutentha kwa chipinda kwa maola awiri, sambitsani mbaleyo ndi 0.05% Tween-20/PBS (wash buffer) kwa masekondi 3, zitsanzo za CSF ndi plasma zinawonjezedwa ku zitsime zomwe zinasungunuka ndi blocking buffer (plasma 1:10,000, CSF 1:115). Kenako mbaleyo inayikidwa mu incubation usiku wonse pa 4°C ndi detection antibody (1 μg/ml, anti-streptavidin-HRP, Novus NB120-7239). Pambuyo pa masitepe atatu otsukira, streptavidin inapezeka mwa kuyikamo mu yankho la TMB substrate (Roche) kwa mphindi 20. Mukasiya kupanga mtundu ndi 1M H2SO4, yesani kuyamwa kwa utoto pa 450 nm.
Ntchito ya streptavidin-peptide-BACE1 inhibitor complex inayesedwa ndi Aβ(1-40) ELISA malinga ndi njira ya wopanga (Wako, 294-64701). Mwachidule, zitsanzo za CSF zinachepetsedwa mu diluent yokhazikika (1:23) ndipo zinayikidwa usiku wonse pa 4°C m'mabolo a 96-well plates okhala ndi BNT77 capture antibody. Pambuyo pa masitepe asanu otsukira, antibody ya HRP-conjugated BA27 inawonjezedwa ndikuyikidwa kwa maola awiri pa 4°C., kutsatiridwa ndi masitepe asanu otsukira. Aβ(1–40) inapezeka mwa kuikidwa mu yankho la TMB kwa mphindi 30 kutentha kwa chipinda. Pambuyo poti mtundu wa khungu wayimitsidwa ndi yankho loyimitsa, yesani kuyamwa pa 450 nm. Zitsanzo za plasma zinayikidwa mu solid phase extraction isanafike Aβ(1–40) ELISA. Plasma inawonjezedwa ku 0.2% DEA (Sigma) m'mabolo a 96-well plates ndipo inayikidwa kutentha kwa chipinda kwa mphindi 30. Pambuyo potsuka mbale za SPE (Oasis, 186000679) motsatizana ndi madzi ndi 100% methanol, zitsanzo za plasma zinawonjezedwa ku mbale za SPE ndipo madzi onse anachotsedwa. Zitsanzo zinatsukidwa (choyamba ndi 5% methanol kenako 30% methanol) ndikuchotsedwa ndi 2% NH4OH/90% methanol. Pambuyo poumitsa eluate pa 55°C kwa mphindi 99 pa mphamvu ya N2 yokhazikika, zitsanzo zinachepetsedwa mu zosungunulira zokhazikika ndipo Aβ(1–40) inayesedwa monga tafotokozera pamwambapa.
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