Ihuriro ryo gukingira amatungo

Inkomoko y’inyamaswa za Alpha Lifetech zirasobanutse, hamwe n’impushya zo kugenzura no gushyira mu kato, imiterere y’imiterere y’imiterere y’imiterere, inshuro nyinshi zo gukingira, hamwe n’ibisobanuro birambuye byerekana intera ikingira, bitanga serivisi zoroshye zo gukora antibody. Ikingira cyane cyane inyamaswa zo muri Camelidae (Alpaca Liama, n'ingamiya), ishyiraho umusingi wo gukora nanobody. Byongeye kandi, irashobora kandi gukingira inyamaswa zisanzwe (nka Nouvelle-Zélande inkwavu zera, imbeba, n’izindi nyamaswa) ukurikije inshuro zisabwa zo gukingirwa hamwe n’ibisabwa by’ubudahangarwa bw’abakiriya, kubikuramo no kubitandukanya na PBMCs, no kubikoresha mu bushakashatsi bwakurikiyeho (nka antibody ya monoclonal, antibody ya polyclone, nibindi). Abakiriya barashobora gutanga ubudahangarwa bwabo, kandi tuzabagenzura neza. Niba abakiriya batanga immunogène virusi, bakeneye guhagarikwa kandi tuzakora ibizamini byo kudakora kugirango umutekano ubeho. Mugihe kimwe, turashobora kandi guhitamo immunogène ijyanye nibyo abakiriya bakeneye.

Intangiriro yo gukingira inyamaswa

Iyo utanga antibodiyide zikomoka ku nyamaswa nka antibodiyite za monoclonal, antibodiyite ya polyclone, na nanobodies, intambwe yambere ni ugukingira inyamaswa no kubona selile B, antibodies, cyangwa PBMCs kugirango zitegure kubyara umusaruro wa antibody, guteza imbere ubushakashatsi bwibanze bwa siyansi, guteza imbere ibikoresho byo gusuzuma indwara, no koroshya kuvumbura no guteza imbere imiti mishya. Ingamiya, alpacas, Liama, nizindi ngamiya muri Camelidae zirashobora kubyara amasomero ya nanobody (antibody imwe ya domaine) binyuze mu gukusanya amaraso no gukuramo PBMCs. Inkwavu n’izindi nyamaswa zirashobora gukingirwa kandi antibodies zirashobora gukurwa mumibiri yabo. Nyuma yo kwezwa, zirashobora gukoreshwa mugukora antibodiyite za polyclone. Imbeba, imbeba, nibindi birashobora gukoreshwa mugukora antibodiyite za monoclonal muguhuza ingirabuzimafatizo B yakuwe hamwe na selile myeloma. Mbere yo gukingirwa, hakwiye kwitabwaho ku mahame ya 3R (gusimbuza, kugabanya, gutunganya), bishobora kugabanya cyane umubare w’inyamaswa zikoreshwa ku buryo bukenewe, kunoza imikorere y’ubushakashatsi, no kugabanya ububabare bw’inyamaswa. Igishushanyo mbonera gikwiye, guhitamo antibodi zikwiye, hamwe nuburyo bukomeye bwo kwemeza nabyo bigomba gukurikizwa.

Gutondekanya immunogène - umusaruro wa nanobody

Ubwoko bwa Immunogens	Urugero	Gutegura Immunogen	Inyandiko
Poroteyine Immunogens	Enzymes, proteyine, uburozi bwa bagiteri, nibindi bintu	Hitamo uburyo butandukanye bwo kwerekana poroteyine: E. coli, umusemburo, udukoko twangiza, inyamaswa z’inyamabere, na sisitemu idafite selile	Witondere ibintu nkubushyuhe, igihe, pH, hamwe numubiri ushiramo bigira ingaruka kumagambo ya poroteyine
Nucleic Acide Immunogens	ADN, RNA	ADN Immunogen: Kubaka ADN ya plasmide, clone ikwirakwiza imvugo ikwiranye na genes zigenewe, ingirabuzimafatizo z'umuco kugirango yongere ADN ya plasmide, kandi ikuremo ADN ifite isuku nyinshi. RNA Immunogen: Ukeneye kongeramo Imiterere yingofero n'umurizo wa PolyA kugirango wirinde gutesha agaciro	Mugihe utegura immunogène ya ADN, ugomba kwitondera ingirabuzimafatizo zibakira, kandi mugihe utegura immunogène ya RNA, hakwiye kwitabwaho kugirango hirindwe kwangirika kwa RNA.
Immunogens ya virusi	Virusi yose idakingiye urukingo, urukingo rwa subunit, urukingo rwa virusi ya virusi (adenovirus, lentivirus, nibindi), urukingo rwa mRAN.	Mugihe utegura urukingo rwose rukingiwe urukingo, virusi ibanza kudakora kugirango umutekano wiyongere. Kwipimisha uburozi bikoreshwa mukumenya niba kudakora byuzuye. Inkingo za Subunit zisaba gusa poroteyine zo hejuru za virusi nka antigene kandi zigomba gutekereza ku mikorere y’ubudahangarwa.	Adjuvants igomba kongerwaho kugirango itange ingaruka z'umubiri, hakwiye kwitabwaho virusi igomba kuba idakora, hakenewe titerite ya virusi ikwiye kugirango habeho ingaruka z'umubiri, kandi umutekano wibikorwa bigomba gutegurwa cyane.

Gukingira amatungo cyangwa igihe

Guhitamo Amatungo no Gutegura Antigen

Guhitamo inyamaswa: Hitamo alpacas yimyaka iboneye na physique nzima mumatsinda meza, hanyuma ukore isuzuma ryamaraso no gusuzuma indwara kugirango urebe ko nta ndwara zandura cyangwa ibindi bibazo byubuzima.

Gutegura immunogene: Immunogène nurufunguzo rwo gukingira indwara kandi bisaba guhitamo poroteyine zikwiye, peptide, cyangwa ibindi bintu bikwiye bishingiye kubisabwa na antibody.

Alpaca irashobora gukingirwa hamwe na antigene 1-3 icyarimwe, hamwe na antigen yose hamwe ya 1-2mg kuri buri gukingirwa hamwe nubunini buri munsi ya 2mL. Mbere yo gukingirwa, antigen hamwe na aduvant byatewe mu kigero cya 1: 1 kugirango bibe imvange imwe, ibikwa kuri 4 ℃.

Alpaca ikingira

Andika numero yamatwi ya alpaca hanyuma utangire igeragezwa ryikingira. Injira muri lymph node hafi yijosi rya alpaca kumpande zombi, ufite amanota 2 kuruhande kandi hafi 0.4mL ya antigen emulisile yatewe kuri buri ngingo. Nyuma yo gukingirwa, reba igice cyisaha kugirango wemeze ko alpaca idafite ibimenyetso bitameze neza. Gukingira bigomba gutangwa buri byumweru 2, byibuze byibuze 4.

Gukusanya amaraso

Nyuma yo gukingirwa 4 muminsi 5-7, kusanya 50ml yamaraso mumitsi yijosi rya alpaca.

Gutandukanya serumu

Amaraso yakusanyirijwe hamwe kugirango asuzume ubudahangarwa mbere yo gukingira antigen, hamwe na 5mL yamaraso yafashwe buri gihe; Kuri uwo munsi, amaraso yashyizwe kuri 400 xg mu minota 30 ukoresheje 25 ℃ centrifuge yabanje gukonja. Igice cyo hejuru cya serumu cyatandukanijwe kandi kibikwa kugirango ikurikirane antibody titer.

Gutandukanya lymphocytes

Banza wongere 15mL yumuti utandukanya selile, hanyuma wongereho buhoro 15mL yamaraso kumuyoboro wa 50mL centrifuge. Witondere kandi utinde mugihe wongeyeho amaraso kugirango wirinde kuvanga amaraso nigisubizo cyo gutandukana. Banza ukonje centrifuge kuri 25 ℃, centrifuge kuri 400 xg muminota 30, hanyuma ubike serumu yo hejuru mumiyoboro mishya ya centrifuge kuri -80 ℃; Koresha umuyoboro wifuza ipamba yo hagati igizwe na selile yo hejuru yumubiri mumyanya mishya ya 50ml centrifuge. Ongeramo 10mL PBS buffer mubushyuhe bwicyumba kuri buri tube, centrifuge kuri 25 ℃ na 400 xg muminota 20. Kuraho ndengakamere, ongeramo 5mL ya buffer ya PBS mubushyuhe bwicyumba kuri buri tube, uvange witonze, ubare umubare wingirabuzimafatizo ukoresheje hemocytometero, hanyuma centrifuge kuri 25 ℃ na 400 xg muminota 20. Kuraho ndengakamere hanyuma ushonga lymphocytes zabonywe na RNAiso Plus ukurikije umubare w'utugingo ngengabuzima kugirango tubone lysate ya selile 10 ^ 7 / mL, ibikwa kuri -80 ℃.

Igishushanyo 1: Igishushanyo mbonera cyerekana ingamba zitandukanye zo kuvumbura Antibody hamwe nicyiciro cyubushakashatsi kirimo. (Inkomoko y'ishusho: Laustsen, Andreas H. n'abandi.)

Abakiriya batanga immunogens-igenzura ubuziranenge

Ubwoko bwa Immunogens	Ibisabwa	Uburyo bwo kugenzura ubuziranenge
Peptide / Ingero ntoya ya molekile	Imiterere yo gusenyuka (cyane cyane bitewe nuburyo izakora fage), imiterere ya molekile / urutonde rwa peptide, raporo ya synthesis (HPLC / MS / HNMR)	Kugaragaza HPLC / MS
Ingero za poroteyine	SDS-PAGE / WB, imiterere yo kongera kwiyubaka, buffer, amakuru yikirango, umubare wuzuye (20-50ug), kwibanda, kweza, nibindi	SDS-URUPAPURO / WB
Ingero z'akagari	Ubwoko bwakagari (selile primaire / gene yahinduwe selile), imiterere yutugari (gushya / gukonjeshwa gushya), ubwoko bwikura (adherent / guhagarikwa), ibisabwa hagati yumuco, umubare wutugari twatanzwe, umubare wutugingo twatanzwe, ingirabuzimafatizo zahinduwe kugirango tumenye neza intego zo gusuzuma (abakiriya bahuye na antibody yibanze), ingirabuzimafatizo zikonje kugirango hamenyekane igipimo cyo gukira	Ahanini yibasiye ingirabuzimafatizo zahinduwe, ukoresheje abakiriya batanze antibodi yibanze kugirango ELISA imenyekanishe intego (isosiyete yacu itanga antibodiyumu ya kabiri ya HRP), gutahura cytometrie (isosiyete yacu irashobora gutanga antibodiyumu ya kabiri ya fluorescent).

Ni ubuhe buryo bukoreshwa mu gukingira inyamaswa?

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Antibodiyite za polyclone zirashobora gukorwa no gukingira inyamaswa, antibodiyite za monoclonal zirashobora gukorwa muguhuza selile B hamwe na selile myeloma, kandi PBMCs irashobora gukurwa mumasoko y'ingamiya (alpaca, ingamiya, alpaca, nibindi) kugirango ikore ibice bitandukanye bya antibody nka nanobodies, ibice bya Fab, scFv antibody, nibindi.
Ni ubuhe buryo bwo kwirinda ubudahangarwa bw'inyamaswa?

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Kugirango habeho nanoantibodies yinyamaswa zikomoka ku ngamiya, birakenewe gukingirwa 5-6. Ingero zamaraso zigomba gukusanywa nkigenzura mbere yo gukingirwa, hagati yicyumweru 2 hagati ya buri rukingo. Nyuma yo gukingirwa kwa kane, amaraso meza n'amaraso mabi adakingiwe bigomba gukusanywa kugirango ELISA imenye titer. Niba titer iri munsi ya 10 ^ 6, umubare winkingo ugomba kwiyongera kugeza igihe titer igeze kurwego rukwiye. Kubindi bikingira inyamaswa nko gukingira urukwavu (urwego rwiza cyangwa rusanzwe rwa poroteyine ya antigen yo gukingira urukwavu ni 50-1000ug), gukingira imbeba (urwego rwiza cyangwa rusanzwe rwa poroteyine antigen yo gukingira imbeba ni 5-50ug, bisaba selile 10 ^ 6 niba ari selile, na 10-50ug kuri acide nucleic cyangwa karubone), gukingira ihene ni byiza kuri 500- dosiye, umutekano, hamwe na efficacy ya immunogène.
Nigute ushobora guhitamo inyamaswa zikingira?

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Ukurikije ubwoko bwa antibody isabwa nabakiriya, niba ukeneye kubyara nanobodies cyangwa ibindi bice bya antibody, urashobora guhitamo inyamaswa zikomoka ku ngamiya (ingamiya, alpaka, alpaka) kugirango ukingire. Niba ukeneye gukora antibodiyite za monoclonal cyangwa polyclone, ugomba guhitamo ubwoko bujyanye ukurikije ingano ya serumu, antigen, na antigen. Umubare ntarengwa wa serumu ku nkwavu ni 500ml, bisaba antigen nkeya kandi niwo mwanya wambere wo gukora antibodiyite za polyclone. Umubare ntarengwa wa serumu ku mbeba ni 2ml, naho serumu ntarengwa ku mbeba ni 20ml, ubusanzwe ni bwo buryo bwiza bwo guhitamo antibodiyite za monoclonal. Ihene yakozwe na antibodies zifitanye isano ryinshi, kandi Alpha Lifetech irashobora kugena gahunda yihariye yo gukingira kuri wewe ukurikije ibyo usabwa.
Antigene ikeneye gukoreshwa hamwe na adjuvant? Nigute ushobora guhitamo?

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Hariho ubwoko bubiri bwinyongera: bushingiye kumavuta namazi. Gukoresha mu buryo bushyize mu gaciro ni ngombwa mu gutera antibody ikomeye kuri antigene zishonga. Irekurwa rirambye ryimiti isobanura ko hashobora gukoreshwa dosiye nkeya ya antigene, kandi ibisubizo bya antibody biraramba. Urushinge rwa mbere rugomba gukoreshwa hamwe na adjuvant. Mubisanzwe birasabwa gukoresha imiti ya Freund mugihe ingano ya immunogen ari nto.
Kwirinda gukingira alpaca?

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(1) Guhitamo alpaca na antigen yubudahangarwa nurufunguzo rwo gukingira neza. Guhitamo ingamiya igereranijwe neza ntabwo ari byiza. Isuku noguhindura neza antigene yumubiri ningirakamaro mugusuzuma antibodi zibereye zikoreshwa nyuma yo gukingirwa na alpacas. Ubuziranenge bwa poroteyine antigene muri rusange ntabwo buri munsi ya 90%.

(2) Gutandukanya lymphocytes: Gutandukanya ingirabuzimafatizo ku gihe birashobora gukumira neza hemolysis nyuma yo gukusanya amaraso kugirango bigerweho neza.

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yigenga yatezimbere

Imikorere idasanzwe ya formulaire yatunganijwe neza kuri sisitemu yubudahangarwa ya alpaca, itanga imbaraga zidasanzwe kandi zidasanzwe kuruta imiti gakondo.

Gutanga inkingo
bisanzwe

Turasezeranye kurangiza gukingira mugihe cyagenwe, gutanga serumu ndende, no kwandika buri ntambwe binyuze muri raporo zirambuye.

umushinga Kugenzura ubuziranenge

Buri mushinga uhura nuburyo bunoze bwo kugenzura ubuziranenge kugirango harebwe itangwa rya antibodies zujuje ubuziranenge kubakiriya.

biragaragaraAlpacaAmavu n'amavuko

Dufite abaturage ba alpaca nzima itanga inkunga muri gahunda zitandukanye zo gukingira.

Niba ufite ikibazo, nyamuneka twandikire igihe icyo aricyo cyose.

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