SARS-COV-2 nruab nrab tshuaj tiv thaiv antibldy nrhiav cov khoom siv (Elisa)
【Lub ntsiab lus】
Lub SARS-COV-2 Meshrationalizing tshuaj tiv thaiv tshuaj tiv thaiv cov khoom siv yog raws cov elisa vib this.
Siv huv receptor khi sau npe (RBD), protein los ntawm tus kab mob viral spike (s) protein ntau thiab tus tswv tsev
Receptor Ace2, qhov kev ntsuas no yog tsim los ua kom Mimic tus kab mob-host nruab nrab sib cuam tshuam.
Cov tshuaj calibrators, cov tswj hwm zoo, thiab ntshav ntshav lossis ntshav ntshav lossis ntshav cov qauv ntsuas yog cov kev sib xyaw kom zoo
Buffer uas muaj HACE2-HRP conjugate aliquoted nyob rau hauv me me hlab. Tom qab ntawd cov mixtures tau pauv hauv
Cov Hluav Taws Xob Microplate muaj cov immobilized recombant regbant-cov-2 rbd tawg (RBD) rau
tsim kom loj hlob. Thaum 30-feeb tsim kom loj hlob, RBD cov tshuaj tiv thaiv tshwj xeeb hauv cov tshuaj ntsuas, QC thiab
Cov qauv yuav sib tw nrog HACE2-HRP rau tshwj xeeb khi RBD immobilized hauv cov dej. Tom qab
Qhov tsim kom loj hlob, cov dej no tau ntxuav tau 4 zaug tshem tawm Unbound HACE2-HRP conjugate. Kev daws teeb meem ntawm
TMB yog tom qab ntawd ntxiv thiab muab ncu rau 20 feeb ntawm chav sov, ua rau muaj kev txhim kho a
xiav xim. Cov kev txhim kho cov xim tau nres nrog kev sib ntxiv ntawm 1n hcl, thiab nqus tau yog
ntsuas ntsuas spectrophottometrically ntawm 450 nm. Qhov kev siv ntawm cov xim tsim yog sib npaug rau cov
Tus nqi ntawm enzyme tam sim no, thiab yog inversely ntsig txog tus nqi ntawm cov qauv assayed nyob rau tib txoj kev.
Los ntawm kev sib piv nrog txoj kev ntsuas hluav taws xob tau tsim los ntawm cov calibrators tau muab los ntawm cov calibrators, kev sib haum xeeb ntawm
nruab nrab cov tshuaj tiv thaiv kab mob hauv cov qauv tsis paub yog xam tau.
【Cov ntaub ntawv yuav tsum tau tab sis tsis muab】
1. Distilled lossis deionized dej
2. Precision pipettes: 10μL, 100μl, 200μL thiab 1 ml
3. Cov Lus Qhia Kav Tos Pipette
4. Microplate Reader Muaj peev xwm ntawm kev nyeem ntawv ntawm 450nm.
5. Absoster
6. Daim Ntawv Xov Xwm
7. Vortex tov khoom lossis sib npaug
【Sau cov ntawv sau cia thiab cia】
1. Ntshav qab thiab ntshav thiab cov hnoos qeev uas muaj k2-edta tuaj yeem siv rau cov khoom siv no.
2. Cov quav yuav tsum capped thiab tej zaum yuav khaws cia txog 48 teev ntawm 28 teev ntawm 2 ° C - 8 ° C uantej assaying.
Cov zis ntim tau ua rau lub sijhawm ntev dua (txog 6 lub hlis) yuav tsum khov tsuas yog ib zaug ntawm -20 ° C CE ua ntej muaj txiaj ntsig.
Tsis txhob rov ua kom khov-thaw mus.
Raws tu raws tu raws tu txheej
【Reagent npaj】
1. Txhua reagents yuav tsum tau coj tawm ntawm tub yees thiab tau tso cai rov qab mus rau chav sov ua ntej siv
(20 ° rau 25 ° C). Txuag tag nrho cov reagents hauv tub yees kom sai tom qab siv.
2. Txhua qhov piv txwv thiab kev tswj yuav tsum yog vortexed ua ntej siv.
3. HACE2-HRP Txuj Ci Npaj: Dilute HVE2-HRP mloog zoo ntawm 1: 51 Dilution Ratio nrog Dilight
Tsis. Piv txwv li, dilute 100 μl ntawm HACE2-HRP mloog zoo nrog 5.0mL ntawm HRP dilution tsis
Ua ib txoj kev daws teeb meem HACE2-HRP.
4. 1 × Ntxuav cov tshuaj ua kom haum: Dilute 20 × ntxuav daws nrog cov dej uas muaj kev nplua nuj lossis dej distilled nrog a
Ntim piv txwv ntawm 1:19. Piv txwv, dilute 20 ml ntawm 20 × ntxuav nrog 380 ml ntawm deionized lossis
Distilled dej kom ua 400 ml ntawm 1 × ntxuav cov tshuaj.
【Cov txheej txheem ntsuas】
1. Hauv kev cais tub, aliquot 120μl ntawm npaj HACE2-HRP daws.
2. Ntxiv 6 μl ntawm cov ntsuas kub, cov qauv tsis paub, tswj tau zoo hauv txhua lub raj thiab sib tov zoo.
3. Hloov 100μL ntawm txhua qhov sib xyaw npaj rau theem 2 rau hauv kev sib piv microplate zoo raws li
txhawm rau ua ntej kev ntsuas kev ntsuas.
3. Npog lub phaj nrog phaj foob thiab tsim ntawm 37 ° C rau 30 feeb.
4. Tshem cov phaj ntim thiab ntxuav cov phaj nrog kwv yees li 300 μl ntawm 1 × ntxuav daws ntawm plaub lub sij hawm.
5. Coj mus rhaub cov phaj rau ntawm daim ntawv phuam kom tshem cov kua dej uas seem hauv cov dej hauv cov kauj ruam tom qab cov kauj ruam ntxuav.
6. Ntxiv 100 μl ntawm TMB kev daws rau txhua qhov dej thiab ua kom lub phaj tsaus ntuj ntawm 20 - 25 ° C rau 20 feeb.
7. Ntxiv 50 μl ntawm kev daws teeb meem rau txhua qhov zoo kom nres qhov tshuaj tiv thaiv.
8. Nyeem cov pa ntawm micpplate nyeem ntawv ntawm 450 nm tsis pub dhau 10 feeb (630nm raws li accessory yog
Pom zoo rau cov kev ua tau zoo dua).