Ihuriro ryo gupima

Ukurikije urubuga rwa Octet na Biacore-T2000, Alpha Lifetech irashobora gutanga serivisi zizewe zifatika. Turashobora gutanga ibyemezo byerekana urugero rwinshi, nka antibodies, selile, proteyine, molekile ntoya nibindi. Hariho uburyo butandukanye bwo kumenya isano, harimo plasmon resonance yo hejuru (SPR) hamwe no guhuza bio-layer (BLI) kugena isano.

Alpha Lifetech ifite urubuga rwumwuga kandi rwukuri rugena isano ihuza ELISA no kwiyemeza gukundana, itanga ibisubizo byumwuga, bikora neza, byihuse, kandi byukuri kubisubizo byabakiriya kwisi yose. Turashobora gutanga uburyo bubiri kubakiriya bahitamo: Kumenyekanisha Byihuse Byihuse no Kumenya neza. Kwiyemeza kwihuta ni icyemezo cyo guhuriza hamwe, mugihe kugena neza neza bishobora gupima isano yibitekerezo bitandukanye. Byakoreshejwe mubushakashatsi bwimikoranire hagati ya molekile ntoya itandukanye, peptide, proteyine, oligonucleotide, na oligomers, hamwe na lipide, bacteriofage, virusi, na selile. Ihuriro ryo gupima affinity rishobora gushiraho urufatiro rwo gupima ibiyobyabwenge no gusuzuma, kuvumbura antibody, no koroshya ubushakashatsi.

Intangiriro yo Guhuza

Ubusabane ni ngombwa mugusuzuma imikoranire hagati yimiterere, imiti yibiyobyabwenge, no gusuzuma ibiyobyabwenge. Imikoranire hagati yimikorere irashobora gusobanurwa nuburinganire: L + R = LR, aho L igereranya ligande yubuntu, R igereranya reseptors idahuza, naho LR igereranya ligand-reseptor ihuza imipaka. Guhuza ibisubizo bisobanura imikoranire hagati yimikorere, aho guhanahana imbaraga bibaho hagati yimipaka nimbibi mugihe cyibisubizo kugeza uburinganire bugeze. Ibi birashobora gusobanurwa nibipimo bibiri bihagaze, Kon (guhuza igipimo gihoraho) na Koff (igipimo cyo gutandukana gihoraho), cya reaction. Agaciro Kd, nigisubizo cyo guhuza guhoraho (Ka), ni Koff / Kon, ikintu cyingenzi gihoraho kubitekerezo. Kubwibyo, gukomera guhuza molekile ebyiri, niko bigenda byiyongera. Gutoya Kd agaciro, ibinyuranye. Iri gereranya rishobora kugaragazwa nkumugozi wa S-shusho kumurongo wigice cya logarithmic, hamwe na ligand yibanze kuri x-axis (kuri logarithmic scale axis) nimbibi zigabanywa kuri y-axis. Umurongo ucagaguye ugereranya ligand yibanze kuri Kd (1 nM) ya 0.5 ihuza igice.

Igishushanyo 1: Sigmoidal ihuza umurongo wuburyo butandukanye bwa ligand ihujwe na reseptor selile. (Inkomoko yerekana: Umuhigi SA, Cochran JR. Utugingo ngengabuzima twa selile kugirango tumenye isano ya poroteyine-poroteyine: Ikoranabuhanga no gutekereza.)

Uburyo bwo kumenya isano

ELISA ihuza isano

Tekiniki ikoreshwa cyane mukwiga antibody ifitanye isano nuburyo bwa ELISA, burangwa nuburyo bworoshye, umuvuduko, ubworoherane, ibyiyumvo byinshi, kandi byihariye. Irashobora gukoresha urugero ruke rwa reagent (ni ukuvuga antibodies na antigene) kandi igapima isano ya antibody idakeneye reagent yo kweza. Muguhagarika antigen hejuru yubutaka no kuyimenya ukoresheje antibody yibanze, antibody yanditseho kabiri ikora hamwe na antibody yibanze kugirango isome kandi isesengure amakuru mumasomo ya immunosorbent asoma (ELISA) umusomyi.

Igishushanyo 2: ELISA isa nubushakashatsi bwo gusuzuma guhuza peptide yabugenewe kubyo bagenewe. (Inkomoko yerekana: Hajikarimlou, Maryam, n'abandi, 2022. Uburyo bwo kubara bwo gukora peptide yihuse yerekana proteine ya SARS-CoV-2 S.)

Ubuso bwa Plasmon Resonance (SPR) guhuza isano

Ikoranabuhanga rya SPR ryerekana cyane cyane impinduka zerekana. Hifashishijwe ibintu bisanzwe bya optique hamwe na resonance phenomenon yumucyo, tekinoroji yo gusesengura biosensing kugirango imikoranire hagati ya biomolecules irashobora kubakwa kugirango hamenyekane imikoranire hagati ya ligande na analyite kuri chip biosensing. Ibimenyetso byihariye byo guhuza no gukorana hagati ya biomolecules birashobora kuboneka mugukurikirana impinduka zikomeye mumpande za SPR mugihe cyibinyabuzima.

Igishushanyo 3: Isesengura rya plasmon resonance (SPR) isesengura rya H10 / AGR2 guhuza. (Inkomoko yerekana: Garri, Carolina, n'abandi, 2018. Kumenyekanisha, kuranga, no gukoresha peptide nshya irwanya icyiciro cya mbere cya homologi 2 (AGR2).)

Kwivanga kwa Bio (BLI) guhuza isano

Ikoreshwa rya tekinoroji ya Biofilm ni ikirango kitarangwamo ikirango, igihe nyacyo cyo kugenzura optique yo gutahura ikoreshwa cyane cyane mu gusesengura ingano yuzuye y’imikoranire hagati y’imikoranire hagati ya poroteyine. Ubu buhanga bukoresha biosensor ishingiye kuri probe kugirango hamenyekane neza impinduka mubyimbye bya biofilm kuri sample. Mugutahura impinduka zo kwimuka kwa interineti, guhuza no gutandukana hagati ya biomolecules zikorana hejuru ya sensor iramenyekana, kandi igihe nyacyo cyo kwimura (nm) cyerekana intera igaragara.

Igishushanyo 4: Biolayer interferometrie (BLI) isuzuma hagati ya ALDRG na chitinoligosaccharide. (Inkomoko yerekana: Li, Bing, 2023. Ibimenyetso biranga Transcriptome igereranya hagati ya Rhizomorphs na Hyphae ya Armillariya sp. 541 Kugira uruhare muri Symbiose ya Fungal ushimangira kuri LysM.)

Kugereranya tekinoroji ya BLI na SPR

Izina ry'ikoranabuhanga	BLI (Interineti ya Bio-Layeri)	SPR (Ubuso bwa Plasmon Resonance)
Ihame	Gupima impinduka muburyo bwo gutandukanya urumuri rwerekanwe hejuru ya sensor, kumenya imikoranire ya molekile ikoresheje impinduka mubyimbye bya optique kuri bio-layer. Itanga igihe-nyacyo cyo guhuza umurongo (gupima neza).	Gupima imikoranire ya molekulari muguhitamo ibimenyetso byerekana ibimenyetso byangiritse hafi yubuso bwa sensor chip (bibaho iyo urumuri ruhuye na zahabu nikirahure, bigatera impinduka mubyerekana). Amakuru agaragara nkimpinduka muburyo bwa resonance (gupima indirect).
Uruganda	Sartorius	GE
Igikoresho	ForteBio Biosensors	Fungura igikoresho cya SPR
Sisitemu	Sisitemu ya ForteBio Octet (yo gusesengura imikoranire ya molekile)	TraceDrawer (yakozwe na Ridgeview Instruments, Suwede)
Ibyiza	1. Icyitegererezo cyagutse gihuza, gihamye cyane, cyane cyane mugushakisha molekile nto (ibisabwa byihariye kubisuku byintangarugero nibisabwa ntibikomeye). Chip ya SSA irahenze cyane kubushakashatsi. 2. Kwinjira byihuse nigihe gito cyubushakashatsi ugereranije na SPR.	1. Amateka maremare yiterambere, atanga sensibilité yo hejuru ugereranije na BLI. 2. Ibisobanuro binini kandi bikomeye kubikorwa byihariye, nko kumenya poroteyine zidasanzwe cyangwa zifite agaciro gakomeye hamwe namakuru yihariye.
Ibibi	1. Ibisobanuro byukuri biri hasi gato ugereranije na SPR. 2. Bisaba gufata neza ibikoresho. 3. Igiciro cya chip ya SSA kiri hejuru.	1. Buffer conditions zo kumenya molekile nto cyane zirashobora gusaba, byongera ibyago byo kunanirwa gutahura. 2. Chips muri rusange ihenze kuruta izikoreshwa muri BLI. 3. Guhumeka byintangarugero mugihe cyubushakashatsi bwagutse birashobora kuba ikibazo.
Ubwoko bwa Chip	SSA Chip	NTA Chip

Igipimo cyo gupima uburinganire

Kwiyemeza kuba hafi bishobora kuba birimo antigen-antibody (antigen-antibody ikomeye, antigen-antibody idakomeye), protein-protein, protein-peptide, proteine-ntoya, na proteine ADN / RNA (aptamer). Iyo upima KD, birakenewe kumenya imwe murwego rwohejuru. Iyo molekile ntoya ihambiriye, uburemere bumwe bwa molekile ntibushobora kuba munsi ya 150 daltons.

Andika	Umwanya	Kwirinda
1. Antigen-Antibody	10 ^ -6 kugeza 10 ^ -12	Indangagaciro za Kd za antibodies nyinshi ziri murwego rwa 10 ^ -6-10 ^ -7 kugeza 10 ^ -9. Mubisanzwe abantu bemeza ko antibodiyite nyinshi ziri muri 10 ^ -9, mugihe nyinshi. Antibodies ziri muri 10 ^ -12.
2. Poroteyine - Molekile nto	10 ^ -4 kugeza 10 ^ -5	KD ya molekile nto na proteyine ziri hagati ya 10 ^ -4 na 10 ^ -5, mugihe 10 ^ -3 na 10 ^ -7 nibisanzwe kandi ntibishobora kugera kuri 10 ^ -10. Molekile ntoya irashobora kugera kuri 10 ^ -10.
3. Avidin-Biotin	10 ^ -14	Gukundana birashobora guhita byoroha guhuza, kandi streptavidin cyangwa deglycosylated affinity irashobora gukoreshwa.
4. ADN-Poroteyine	10 ^ -8 kugeza 10 ^ -10	ADN nziza kandi yuzuye; witondere gukumira ingaruka za electrophoreis.

Icyitegererezo gisabwa kugirango umuntu agaragaze isano

Icyitegererezo	Ibisabwa
1. Icyitegererezo kinini cya molekile	Poroteyine> 50 µg, antibody> 100 µg, poroteyine ya biotinylated> 200 µg; poroteyine idafite biotine> 2 mg, ibisabwa byera> 90%, igisubizo cya buffer: PBS, HEPBS. Ntushobora kubamo amatsinda ya imidazole; kugenzura ubuziranenge birakenewe.
2. Icyitegererezo gito cya molekile	Umubare> 1mg, ifu cyangwa amazi, amazi agomba gukemuka mumazi cyangwa DMSO. Gerageza kutarimo glycerol, imidazole, trehalose cyangwa indi myunyu; gerageza wirinde reagent hamwe nitsinda rya amino nka Tris muri buffer, muri rusange PBS, HEPPS, nibindi bidafite reagent.

Ibyiza bya platifike yo gupima

Isesengura ryinshi ry'icyitegererezo

Alpha Lifetech irashobora gutanga uburinganire bwintangarugero zitandukanye, harimo antibodies, selile, proteyine, nibindi binyabuzima.

Ihuriro ryikoranabuhanga rikuze

Dufite tekinoroji igezweho nka spr binding assay, bli binding assay, na elisa binding assay.

Guhitamo umushinga woroshye

Abakiriya barashobora guhitamo hagati yo kwiyemeza kwihuta no kugena neza.

Ibisubizo bihanitse

Itsinda ryacu rya tekiniki ryumwuga rirashobora kwemeza neza ibisubizo nyabyo, byukuri, kandi byizewe.

InyigoURUBANZA

BLI yihuta yihuta antibody na aptamer affinity assay

Gufata biotinylated aptamers hamwe na SA probe yihariye ukayishonga. Gabanya kandi ugabanye icyitegererezo kugirango ushire hamwe, ushimangire iperereza ryihariye ryafashwe Target 1-5 aptamers, hanyuma nyuma yo kuzuza ibimenyetso, uhambire kuri sample. Noneho ubyongereze ku isahani 96-iriba.

Igishushanyo 5: Gukwirakwiza imyanya yo gutahura ibyitegererezo 96-byapa. B bivuga kuri buffer ikoreshwa mukuringaniza no gutandukanya sensor. L: Intego ya Biotin 1-5 aptamers, 221: Icyitegererezo.

Witondere gucukura kugirango umenye neza amakuru neza, kandi ushireho gahunda ijyanye no kubona ibisubizo:

Igishushanyo cya 6: Igishushanyo gikwiye igishushanyo hagati ya Target 1, 2, na 3 aptamers hamwe nicyitegererezo. (CH 1 \ 3 \ 5 byerekana ibimenyetso namakuru yimikoranire hagati ya Target 1 \ 2 \ 3 aptamers probe hamwe nicyitegererezo.)

Igishushanyo 7: Igishushanyo gikwiye igishushanyo hagati ya Target ya 4 na 5 aptamers hamwe nicyitegererezo. (CH 1 \ 3 byerekana ibimenyetso namakuru yimikoranire hagati ya Target 4 \ 5 aptamer probe hamwe nicyitegererezo.)

ibisubizo byerekana

Ibisubizo bikurikira byabonetse: Ubusabane bwa Target 1 adapter kuri sample nyuma yo guhuza ni 9.41 ^ -8; isano ya Target 2 adapter kuri sample nyuma yo guhuza ni 8.32 ^ -8; isano ya Target 3 adapter kuri sample nyuma yo guhuza ni 8.64 ^ -8; isano ya Target 4 adapter kuri sample nyuma yo guhuza ni 3.70 ^ -8; isano ya Target 5 adapter kuri sample nyuma yo guhuza ni 3.01 ^ -8.

Niba ufite ikibazo, nyamuneka twandikire igihe icyo aricyo cyose.

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