Urubuga rwo gupima isano

Hashingiwe kuri Octet na Biacore-T2000, Alpha Lifetech ishobora gutanga serivisi zizewe zo kumenya affinity. Dushobora gutanga affinity ku ngero nyinshi, nka antibody, selile, poroteyine, molekile nto n'ibindi. Hari uburyo butandukanye bwo kumenya affinity, harimo surface plasmon resonance (SPR) na bio-layer interference (BLI) affinity determining.

Alpha Lifetech ifite urubuga rw’ubuhanga kandi rw’ukuri rwo kumenya affinity ruhuza ELISA n’igenamiterere ry’affinity, rutanga ibisubizo by’ubuhanga, byiza, byihuse kandi bifatika ku bakiriya bo ku isi yose. Dushobora gutanga uburyo bubiri abakiriya bashobora guhitamo: Gupima Affinity Byihuse na Precise Affinity Determination. Gupima affinity Byihuse ni ugupima icyiciro kimwe, mu gihe kumenya affinity byihuse bishobora gupima affinity y’ibipimo bitandukanye. Bikoreshwa mu kwiga imikoranire hagati y’ibintu bitandukanye bito bya molekile, peptides, poroteyine, oligonucleotides, na oligomers, hamwe na lipide, bacteriophages, virusi, n’uturemangingo. Urubuga rwo gupima affinity rushobora gushyiraho urufatiro rwo gupima no gusuzuma imiti ya affinity, kuvumbura antibody, no koroshya ubushakashatsi bukurikiraho.

Intangiriro yo guhuza ibintu

Ubwumvikane ni ingenzi mu gusuzuma imikoranire hagati ya molekyuli, isuzuma ry’imiti ishingiye ku bufatanye, no gusuzuma imiti. Imibanire hagati ya molekyuli ishobora gusobanurwa hakoreshejwe ibipimo: L + R = LR, aho L ihagarariye ligands zigenga, R ihagarariye receptors zidafite aho zibohewe, naho LR ihagarariye complexes za ligand-receptor zishingiye ku bufatanye. Uburyo bwo guhuza busobanura imikoranire hagati ya molekyuli, aho guhinduranya ibintu hagati ya leta zishingiye ku bufatanye n’izidafite aho zibohewe mu gihe cy’inyito kugeza igihe uburinganire bugeze. Ibi bishobora gusobanurwa n’ibipimo bibiri, Kon (binding rate constant) na Koff (dissociation rate constant), by’inyito. Agaciro ka Kd, ari ko gahuza ubwumvikane hagati ya molekile (Ka), ni Koff/Kon, ubwumvikane bw’ingenzi ku bufatanye bwa reaction. Kubwibyo, uko guhuza hagati ya molekile ebyiri gukomeye, niko ubufatanye bwiyongera. Uko agaciro ka Kd kaba gato, ni ko n’ubundi. Iki gipimo gishobora kugaragazwa nk’umugozi umeze nka S kuri graph ya semi-logarithmic, hamwe na ligand concentration kuri x-axis (kuri logarithmic scale axis) n’umupaka wa fractional kuri y-axis. Umurongo urimo uduce duto ugaragaza igipimo cya ligand kuri Kd (1 nM) ya 0.5 bidding fraction.

Igishushanyo cya 1: Inzira yo gufatanya ya Sigmoidal y'ibipimo bitandukanye bya ligand ifatanye na receptor y'ubuso bw'uturemangingo. (Inkomoko y'inyongera: Hunter SA, Cochran JR. Isuzuma ry’imibanire y’uturemangingo mu kumenya isano iri hagati ya poroteyine na poroteyine: Ikoranabuhanga n’ibyo umuntu yakwitaho.)

Uburyo bwo kumenya isano

Isuzuma rya ELISA rijyanye n'ubudahangarwa

Uburyo bukoreshwa cyane mu kwiga uburyo bwo kwirinda indwara ziterwa n’udukoko bushingiye ku buryo bwa ELISA, burangwa no koroherwa, umuvuduko, ubworoherane, ubushobozi bwo kumva neza, no kuba bufite umwihariko ukomeye. Bushobora gukoresha udukoko duke (ni ukuvuga udukoko duto na antigens) no gupima uburyo bwo kwirinda indwara ziterwa n’udukoko bitabaye ngombwa ko hakorwa udukoko two gusukura. Mu guhagarika antigen ku buso bukomeye no kuyimenya hakoreshejwe antibody y’ibanze, antibody y’inyongera iriho izina ikorana n’ antibody y’ibanze kugira ngo isome kandi isesengure amakuru mu gisomwa cya enzyme-linked immunosorbent assay (ELISA).

Igishushanyo cya 2: Isuzuma rimeze nka ELISA kugira ngo harebwe uburyo peptides zakozwe zifatanye n'intego zazo. (Inkomoko y'aho ziherereye: Hajikarimlou, Maryam, n'abandi, 2022. Uburyo bwo kubara bwo gushushanya byihuse peptide zipima poroteyine S yo ku buso bwa SARS-CoV-2.)

Isuzuma ry'ubudahangarwa bwo guhuza plasmon (SPR)

Ikoranabuhanga rya SPR ahanini rimenya impinduka mu gipimo cya refractive. Hifashishijwe ibintu bisanzwe by’urumuri n’ishusho ya resonance y’urumuri, ikoranabuhanga ryo gusesengura biosensing rigamije kumenya imikoranire hagati ya biomolecules n’ibisesengura kuri biosensing chips. Ibimenyetso byihariye byo gufatana no gukorana hagati ya biomolecules bishobora kuboneka binyuze mu kugenzura impinduka zihinduka mu nguni za SPR mu gihe cy’ibikorwa by’ibinyabuzima.

Ishusho ya 3: Isesengura rya resonance ya plasmon yo hejuru (SPR) ku guhuza kwa H10/AGR2. (Inkomoko y'aho iboneka: Garri, Carolina, n'abandi, 2018. Kumenya, imiterere, no gukoresha peptide nshya mu kurwanya homolog 2 y'imbere (AGR2).)

Isuzuma ry’ubudahangarwa bw’ibice bya Bio Layer Interference (BLI)

Ikoranabuhanga rya Biofilm interference ni uburyo bwo gupima optique budafite ikimenyetso, bukoreshwa mu gihe nyacyo, ahanini mu gusesengura imibare y’imikorere ya molekile no kumenya ingano ya poroteyine. Ubu buryo bukoresha biosensor ishingiye kuri probe kugira ngo bumenye neza impinduka mu bunini bwa biofilm ku gipimo. Mu kumenya impinduka mu mihindagurikire ya interference, uburyo bwo gufatana no kwitandukanya hagati ya biomolecules zikorana ku buso bwa sensor buragaragara, kandi uburyo bwo kwimuka mu gihe nyacyo (nm) bwa interference spectrum buragaragazwa.

Igishushanyo cya 4: Isuzuma rya Biolayer interferometry (BLI) hagati ya aLDRG na chitinoligosaccharides. (Inkomoko y'aho: Li, Bing, 2023. Ibimenyetso by'igereranya ry'inyandiko hagati ya Rhizomorphs na Hyphae ya Armillaria sp. 541 Kwitabira ihuriro ry'ibihumyo hibandwa ku miterere ya LysM.)

Kugereranya ikoranabuhanga rya BLI na SPR

Izina ry'ikoranabuhanga	BLI (Bio-Layer Interferometry)	SPR (Uburyo bwo kugarura imiterere y'ubuso bwa Plasmon)
Ihame	Ipima impinduka mu miterere y’urumuri rugaragara ku buso bwa sensor, ikamenya imikoranire ya molekile binyuze mu mpinduka mu bunini bw’urumuri kuri bio-layer. Itanga umurongo wo gufatanya mu gihe nyacyo (igipimo kitaziguye).	Ipima imikoranire ya molekile isuzuma impinduka mu kimenyetso cya refractive index hafi y'ubuso bwa sensor chip (ibaho iyo urumuri rukorana n'umuyoboro wa zahabu n'ikirahure, bigatera impinduka mu kimenyetso cya refractive index). Amakuru agaragara nk'impinduka mu nguni ya resonance (igipimo kitaziguye).
Uruganda	Sartorius	GE
Igikoresho	Ibyuma by'ibinyabuzima bya ForteBio	Igikoresho cya SPR gifunguye
Sisitemu	Sisitemu ya ForteBio Octet (yo gusesengura ikoreshwa rya molekile)	TraceDrawer (yakozwe na Ridgeview Instruments, muri Suwede)
Ibyiza	1. Guhuza ingero mu buryo bugari, ubuziranenge buhebuje, cyane cyane mu kumenya molekile nto (ibisabwa byihariye kugira ngo ingero zisukure n'imiterere yazo ntizigoye cyane). Uduce twa SSA turahendutse mu bushakashatsi bwo gufatanya. 2. Gukoresha neza cyane kandi igihe gito cyo kugerageza ugereranije na SPR.	1. Amateka maremare y'iterambere, atanga ubushobozi bwo kwihuta ugereranije na BLI. 2. Ubuhanga n'ubudahangarwa birushijeho gukoreshwa mu buryo bwihariye, nko kubona poroteyine zidasanzwe cyangwa zifite agaciro gakomeye kandi zifite amakuru yihariye.
Imbogamizi	1. Ubuziranenge bw'amakuru buri hasi gato ugereranije na SPR. 2. Bisaba kubungabunga neza igikoresho. 3. Igiciro cya chip ya SSA kiri hejuru cyane.	1. Imiterere y'uburyo bwo gupima molecule nto cyane ishobora kuba isaba imbaraga nyinshi, bigatuma habaho kunanirwa gupima molecule. 2. Udupira muri rusange turahenze kurusha utwakoreshejwe muri BLI. 3. Ingero z'isuku mu gihe cy'igerageza rirambye zishobora kuba ikibazo.
Ubwoko bwa Chip	Chipu ya SSA	Chipu ya NTA

Igipimo cy'Ubufatanye

Kumenya isano bishobora kuba birimo antigen-antibody (antigen-antibody ikomeye, antigen-antibody idakomeye), poroteyine-poroteyine, poroteyine-peptide, poroteyine-molecule nto, na poroteyine ADN/RNA (aptamer). Mu gupima KD, ni ngombwa kumenya kimwe mu bipimo bya molar. Iyo molekile nto zifatanye, uburemere bumwe bwa molekile ntibushobora kuba munsi ya dalton 150.

Ubwoko	Aho biherereye	Amabwiriza yo Kwirinda
1. Antigen-Antibody	Kuva kuri 10^-6 kugeza kuri 10^-12	Agaciro ka Kd k'uturemangingo twinshi turi hagati ya 10^-6-10^-7 na 10^-9. Muri rusange abantu bemera ko uturemangingo twinshi turi hagati ya 10^-9, mu gihe uturemangingo twinshi turi hagati ya 10^-12.
2. Poroteyine - Molekuli nto	Kuva kuri 10^-4 kugeza kuri 10^-5	KD y'utunyangingo duto na poroteyine iri hagati ya 10^-4 na 10^-5, mu gihe 10^-3 na 10^-7 ari ibisanzwe kandi ntibishobora kugera kuri 10^-10. Molekile nto za Covalent zishobora kugera kuri 10^-10.
3. Avidin-Biotin	10^-14	Ubufatanye bushobora kunyura mu buryo bworoshye ku buryo butari bwihariye, kandi streptavidin cyangwa deglycosylated affinity ishobora gukoreshwa.
4. ADN-Poroteyine	Kuva kuri 10^-8 kugeza kuri 10^-10	ADN yuzuye kandi ifite ireme; witondere kwirinda ingaruka za electrophoresis.

Ibisabwa kugira ngo hamenyekane isano

Urugero	Ibisabwa
1. Urugero rwa Molekuli nini	Poroteyine > 50 µg, antibody > 100 µg, poroteyine ya biotinyled > 200 µg; poroteyine idafite biotin > 2 mg, isabwa ry’ubuziranenge > 90%, umuti wa buffer: PBS, HEPBS. Ntibishobora kuba birimo amatsinda ya imidazole; kugenzura ubuziranenge birakenewe.
2. Urugero rwa Molekuli nto	Ingano >1mg, ifu cyangwa amazi, amazi agomba gushonga mu mazi cyangwa DMSO. Gerageza kutagira glycerol, imidazole, trehalose cyangwa indi myunyu; gerageza kwirinda ibintu bifite amino nka Tris muri buffer, muri rusange PBS, HEPPS, nibindi bidafite amino organic reagents.

Ibyiza byo gupima ihuriro ry'affinity

Isesengura ry'ingero nyinshi

Alpha Lifetech ishobora gutanga isuzuma ry’ubwisungane ku bipimo bitandukanye, birimo abasirikari b’umubiri, uturemangingo, poroteyine, n’izindi biomolecules.

Urubuga rw'ikoranabuhanga rigezweho

Dufite ikoranabuhanga rigezweho nka spr binding assay, bli binding assay, na elisa binding assay.

Guhitamo imishinga ihindagurika

Abakiriya bashobora guhitamo hagati yo kumenya byihuse uburyo umuntu ahagaze cyangwa ahagaze neza.

Ibisubizo by'ukuri cyane

Itsinda ryacu ry’abahanga mu bya tekiniki rishobora kwemeza ibisubizo bifatika, byuzuye kandi byizewe ku bijyanye no kumenya niba umuntu akunda abandi.

Inyigo y'UrubanzaURUBANZA

Isuzuma rya BLI rituma umuntu adashobora kwihuta mu mubiri (antibody) n'isuzuma rya aptamer (aptamer affinity assay) bigabanuka.

Fata aptameri za biotinyled zifite SA probe specificity hanyuma uzishongeshe. Shyira icyitegererezo mu mvange ugishyire mu rugero ruhamye, komeza aptameri zafashwe za Target 1-5, hanyuma nyuma yo gukwirakwiza ikimenyetso, uhambire ku cyitegererezo. Hanyuma uzishyire ku isahani ifite ibiziba 96.

Igishushanyo cya 5: Ikwirakwizwa ry'ahantu ho gupima ingero z'amabati 96. B yerekeza ku gikoresho gikoreshwa mu kuringaniza no gutandukanya sensor. L: Biotin Target 1-5 aptamers, 221: Icyitegererezo.

Witondere gucukumbura kugira ngo urebe neza ko amakuru ahamye kandi ari ukuri, kandi ushyireho gahunda ijyanye nayo kugira ngo ubone ibisubizo:

Igishushanyo cya 6: Imbonerahamwe yo guhuza imikoranire hagati ya aptameri za Target 1, 2, na 3 n'ibipimo. (CH 1 \ 3 \ 5 igaragaza ikimenyetso n'amakuru y'imikoranire hagati ya probe ya aptameri za Target 1 \ 2 \ 3 n'icyitegererezo.)

Ishusho ya 7: Imbonerahamwe yo guhuza imikoranire hagati ya aptameri za Target 4 na 5 n'ibipimo. (CH 1 \ 3 igaragaza ikimenyetso n'amakuru y'imikoranire hagati ya probe ya aptameri ya Target 4 \ 5 ikomeye n'icyitegererezo.)

ibisubizo byerekana

Ibisubizo bikurikira byabonetse: Ingano ya adaptateri ya Target 1 ku cyitegererezo nyuma yo gushyiramo yari 9.41 ^ -8; ingano ya adaptateri ya Target 2 ku cyitegererezo nyuma yo gushyiramo ni 8.32 ^ -8; ingano ya adaptateri ya Target 3 ku cyitegererezo nyuma yo gushyiramo ni 8.64 ^ -8; ingano ya adaptateri ya Target 4 ku cyitegererezo nyuma yo gushyiramo ni 3.70 ^ -8; ingano ya adaptateri ya Target 5 ku cyitegererezo nyuma yo gushyiramo ni 3.01 ^ -8.

Niba ufite ikibazo icyo ari cyo cyose, twandikire igihe icyo ari cyo cyose.

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