I-SARS-CoV-2 Ikhithi yokuQongwa kwe-Antibody engathathi hlangothi (ELISA)
【UMGAQO】
I-SARS-CoV-2 iNeutralizing Antibody Detection Kit isekwe kwindlela ye-ELISA yokhuphiswano.
Ukusebenzisa i-domain binding ehlanjululweyo ye-receptor (RBD), iprotheni evela kwi-viral spike (S) protein kunye ne-host cell
receptor ACE2, olu vavanyo lwenzelwe ukulinganisa i-virus-host neutralizing intsebenziswano.
I-Calibrators, uLawulo loMgangatho, kunye ne-serum okanye iisampulu zeplasma zixutywe kakuhle kwi-dilution
isithinteli esiqulethe i-hACE2-HRP conjugate aliquoted kwiityhubhu ezincinci.Emva koko imixube idluliselwa ngaphakathi
imingxuma yemicroplate equlathe immobilized recombinant SARS-CoV-2 RBD fragment (RBD) for
ekufukameni.Ngexesha le-30-minute incubation, i-antibody ye-RBD ekhethekileyo kwii-calibrators, i-QC kunye
iisampuli ziya kukhuphisana kunye ne-hACE2-HRP ngokubopha ngokuthe ngqo i-RBD engenakunyakaziswa emigodini.Emva koko
i-incubation, imingxuma ihlanjwe amaxesha angama-4 ukususa i-conjugate ye-hACE2-HRP engabotshwanga.Isisombululo se
I-TMB iye yongezwa kwaye ifukanywe imizuzu engama-20 kwiqondo lobushushu begumbi, okukhokelela kuphuhliso lwe-a
umbala oluhlaza.Ukuphuhliswa kombala kumisiwe kunye nokongezwa kwe-1N HCl, kunye ne-absorbence
kulinganiswe i-spectrophotometrically kwi-450 nm.Ubunzulu bombala owenziweyo buhambelana ne
Ubungakanani be-enzyme ekhoyo, kwaye inxulumene ngokungafaniyo nobungakanani bemigangatho evavanyiweyo ngendlela efanayo.
Ngokuthelekisa kunye negophe lokulinganisa elenziwe ngabalinganisi ababonelelweyo, ukugxila kwe
amajoni omzimba neutralizing kwisampulu engaziwayo ke ibalwe.
【IZINTO EZIFUNEKAYO KODWA AKUNIKEZWA】
1. Amanzi adibeneyo okanye ahlanjululweyo
2. Iipayipi ezichanekileyo: 10μL, 100μL, 200μL kunye ne-1 ml
3. Iingcebiso zepipette ezilahlayo
4. Umfundi weMicroplate okwaziyo ukufunda i-absorbence kwi-450nm.
5. Iphepha lokufunxa
6. Iphepha legrafu
7. Umxube weVortex okanye olinganayo
【UKUQOKELELWA KOMSEBENZI NOKUGCINWA】
1. Iisampulu zeSerum kunye nePlasma eziqokelelwe kwiityhubhu eziqulethe i-K2-EDTA zingasetyenziselwa le khithi.
2. Imizekelo kufuneka ifakwe kwi-cap kwaye inokugcinwa ukuya kutsho kwiiyure ezingama-48 kwi-2 °C - 8 °C phambi kovavanyo.
Iisampulu ezigcinwe ixesha elide (ukuya kwiinyanga ezi-6) kufuneka zikhenkcezwe kube kanye kuphela kwi -20 °C phambi kovavanyo.
Kuphephe ukuphindaphinda imijikelo yokunyibilika komkhenkce.
IPROTOCOL
【Ukulungiswa kweReagent】
1. Zonke ii-reagents kufuneka zikhutshwe kwifriji kwaye zivunyelwe ukuba zibuyele kwiqondo lokushisa ngaphambi kokusetyenziswa
(20° ukuya kuma-25°C).Gcina zonke ii-reagents kwifriji ngokukhawuleza emva kokusetyenziswa.
2. Zonke iisampulu kunye nolawulo kufuneka vortexed phambi kokusetyenziswa.
3. I-hACE2-HRP Ukulungiswa kwesisombululo: Dilute i-hACE2-HRP igxininise ku-1: 51 umlinganiselo we-dilution kunye ne-Dilution
Isithinteli.Umzekelo, dilute i-100 μL ye-hACE2-HRP yoxinaniso nge-5.0mL ye-HRP Dilution Buffer ukuya
yenza isisombululo se-hACE2-HRP.
4. 1× Ukuhlamba Isisombululo Ukulungiselela: Nciphisa i-20× yokuhlamba Isisombululo ngamanzi adityanisiweyo okanye adibeneyo
umlinganiselo womthamo we-1:19.Umzekelo, dilute 20 mL of 20× Hlamba Isisombululo nge 380 mL ye deionized okanye
amanzi adibeneyo ukwenza i-400 mL ye-1 × Isisombululo sokuhlamba.
【Inkqubo yoVavanyo】
1. Kwimibhobho eyahlukileyo, i-aliquot 120μL yesisombululo esilungisiweyo se-hACE2-HRP.
2. Yongeza i-6 μL ye-calibrators, iisampuli ezingaziwayo, ulawulo lwekhwalithi kwi-tube nganye kwaye udibanise kakuhle.
3. Dlulisa i-100μL yomxube ngamnye olungiselelwe kwinqanaba lesi-2 kumaqula e-microplate ahambelanayo.
kuqwalaselo lovavanyo oluyilwe kwangaphambili.
3. Gquma ipleyiti ngePlate Sealer uze uyifukamele kwi-37°C imizuzu engama-30.
4. Susa iSitywini sePlayiti uze uhlambe ipleyiti malunga ne-300 μL ye-1× Isicombululo sokuHlamba kwiqula ngalinye amaxesha amane.
5. Cofa ipleyiti kwitawuli yephepha ukususa ulwelo olushiyekileyo equleni emva kokuhlamba amanyathelo.
6. Yongeza i-100 μL ye-TMB Isisombululo kwiqula ngalinye kwaye ufukamele ipleyiti ebumnyameni kwi-20 - 25 ° C imizuzu engama-20.
7. Yongeza i-50 μL yeStop Solution kwiqula ngalinye ukumisa ukusabela.
8. Funda i-absorbence kwi-microplate reader kwi-450 nm ngaphakathi kwemizuzu eyi-10 (630nm njenge-accessory).
kucetyiswa ukusebenza ngokuchanekileyo okuphezulu).
