SARS-CoV-2 Kutabogama Ibikoresho bya Antibody (ELISA)
【IHame】
SARS-CoV-2 Kutabogama Antibody Detection Kit ishingiye kuburyo bwa ELISA burushanwa.
Ukoresheje reseptor isukuye ihuza indangarubuga (RBD), proteyine ziva kuri virusi ya spike (S) na selile yakira
reseptor ACE2, iki kizamini cyagenewe kwigana imikoreshereze ya virusi itabogamye.
Calibator, Igenzura ryiza, hamwe na serumu cyangwa plasma ntangarugero bivanze kugiti cyacyo
buffer irimo HACE2-HRP conjugate yagabanijwe mu tubari duto.Hanyuma imvange yimurwa muri
amariba ya microplate arimo ubudahangarwa bwa recombinant SARS-CoV-2 RBD igice (RBD) kuri
incubation.Mugihe cyiminota 30 yubushakashatsi, RBD antibody yihariye muri kalibatori, QC na
ingero zizarushanwa na HACE2-HRP kugirango zihuze neza RBD yimuwe mumariba.Nyuma
incubation, amariba yogejwe inshuro 4 kugirango akureho HACE2-HRP conjugate.Igisubizo cya
TMB noneho yongeweho kandi ikabikwa muminota 20 mubushyuhe bwicyumba, bikavamo iterambere rya a
ibara ry'ubururu.Iterambere ryamabara rihagarikwa hiyongereyeho 1N HCl, kandi kwinjiza ni
yapimwe spekitifotometometrike kuri 450 nm.Ubwinshi bwamabara yashizweho buringaniye na
ingano ya enzyme ihari, kandi ifitanye isano itandukanye nubunini bwibipimo bisuzumwa muburyo bumwe.
Mugereranije na kalibrasi yumurongo yashizweho na kalibatori yatanzwe, kwibanda kuri
gutesha antibodiyite muri sample itazwi noneho irabaze.
【IBIKORWA BISABWA ARIKO NTIBITANZWE】
1. Amazi yamenetse cyangwa yimana
2. Imiyoboro isobanutse: 10μL, 100μL, 200μL na 1 mL
3. Impanuro zishobora gukoreshwa
4. Umusomyi wa Microplate ashoboye gusoma ibyinjira kuri 450nm.
5. Impapuro zitagaragara
6. Urupapuro
7. Kuvanga Vortex cyangwa bihwanye
【GUKORANYA BIDASANZWE N'UBubiko】
1. Ingero za Serumu na Plasma zegeranijwe mu tubari zirimo K2-EDTA zirashobora gukoreshwa muriki gikoresho.
2. Ibigereranyo bigomba gufatwa kandi birashobora kubikwa mugihe cyamasaha 48 kuri 2 ° C - 8 ° C mbere yo gusuzuma.
Ibigereranyo bifata umwanya muremure (kugeza kumezi 6) bigomba gukonjeshwa rimwe gusa kuri -20 ° C mbere yo gusuzuma.
Irinde gusubiramo inshuro nyinshi.
PROTOKOL
【Gutegura neza】
1. Reagent zose zigomba gukurwa muri firigo hanyuma zikemererwa gusubira mubushyuhe bwicyumba mbere yo kuzikoresha
(20 ° kugeza kuri 25 ° C).Bika reagent zose muri firigo ako kanya nyuma yo kuyikoresha.
2. Ibyitegererezo byose hamwe nubugenzuzi bigomba guhindurwa mbere yo kubikoresha.
3. HACE2-HRP Gutegura Igisubizo: Koresha HACE2-HRP kwibanda kuri 1: 51 igipimo cyo kugabanuka hamwe na Dilution
Buffer.Kurugero, shyira 100 μL ya HACE2-HRP yibanze hamwe na 5.0mL ya HRP Dilution Buffer kugeza
kora igisubizo cya HACE2-HRP.
4. 1.
igipimo cyijwi cya 1:19.Kurugero, shyira mL 20 ya 20 × Gukaraba igisubizo hamwe na 380 mL ya deionised cyangwa
amazi yamenetse kugirango akore 400 mL ya 1 × Gukaraba.
【Uburyo bwo Kwipimisha】
1. Mu tubari dutandukanye, aliquot 120μL yumuti wateguwe HACE2-HRP.
2. Ongeramo 6 μL ya kalibatori, ingero zitazwi, kugenzura ubuziranenge muri buri tube hanyuma uvange neza.
3. Hindura 100μL ya buri mvange yateguwe muntambwe ya 2 mumariba ya microplate ukurikije
Kuri ibizamini byagenwe mbere.
3. Gupfundikira isahani hamwe na Plate Sealer hanyuma ushire kuri 37 ° C muminota 30.
4. Kuraho icyapa cya plaque hanyuma ukarabe isahani hamwe na 300 μL ya 1 × Gukaraba igisubizo kuri iriba inshuro enye.
5. Kanda isahani kumpapuro kugirango ukureho amazi asigaye mumariba nyuma yo koza intambwe.
6. Ongeramo 100 μL yumuti wa TMB kuri buri riba hanyuma ushireho isahani mwijimye kuri 20 - 25 ° C muminota 20.
7. Ongeramo 50 μL yo guhagarika igisubizo kuri buri riba kugirango uhagarike reaction.
8. Soma ibyinjira mubisoma microplate kuri 450 nm muminota 10 (630nm nkuko ibikoresho biri
basabwe gukora neza cyane).
