SARS-CoV-2 Neutralizing Antibody Detection Kit (ELISA)
【PRINCIPLE】
SARS-CoV-2 Neutralizing Antibody Detection Kit yog ua raws li ELISA txoj kev sib tw.
Siv purified receptor binding domain (RBD), protein los ntawm tus kab mob kis kab mob (S) protein thiab cov tswv tsev cell.
receptor ACE2, qhov kev sim no yog tsim los ua kom pom tus kab mob-tus tswv ntawm kev sib cuam tshuam.
Calibrators, Kev Tswj Xyuas Zoo, thiab cov ntshav ntshav los yog cov ntshav plasma yog ib tus neeg sib xyaw ua ke zoo hauv kev dilution
tsis muaj hACE2-HRP conjugate aliquoted hauv cov raj me me.Tom qab ntawd cov mix yog pauv mus rau hauv
microplate wells containing immobilized recombinant SARS-CoV-2 RBD fragment (RBD) for
incubation.Thaum lub sij hawm 30-feeb incubation, RBD cov tshuaj tiv thaiv tshwj xeeb hauv cov calibrators, QC thiab
cov qauv yuav sib tw nrog hACE2-HRP rau kev khi tshwj xeeb ntawm RBD immobilized hauv qhov dej.Tom qab
lub incubation, lub qhov dej yog ntxuav 4 zaug kom tshem tawm unbound hACE2-HRP conjugate.Kev daws ntawm
TMB yog ntxiv thiab incubated rau 20 feeb ntawm chav tsev kub, uas ua rau kev loj hlob ntawm ib tug
xiav xim.Kev txhim kho xim raug nres nrog qhov sib ntxiv ntawm 1N HCl, thiab qhov nqus dej yog
ntsuas spectrophotometrically ntawm 450 nm.Qhov kev siv ntawm cov xim tsim yog proportional rau lub
tus nqi ntawm cov enzyme tam sim no, thiab inversely cuam tshuam rau tus nqi ntawm cov qauv kev soj ntsuam zoo ib yam.
Los ntawm kev sib piv nrog cov calibration nkhaus tsim los ntawm cov muab calibrators, lub concentration ntawm
neutralizing cov tshuaj tiv thaiv kab mob hauv cov qauv tsis paub yog ces suav.
【Cov khoom yuav tsum tau tab sis tsis muab】
1. Distilled los yog deionized dej
2. Precision pipettes: 10μL, 100μL, 200μL thiab 1 mL
3. Cov lus qhia pov tseg pipette
4. Microplate nyeem ntawv muaj peev xwm nyeem ntawv absorbance ntawm 450nm.
5. Daim ntawv nqus
6. Daim duab ntawv
7. Vortex tov khoom lossis sib npaug
【SPECIMEN COLLECTION THIAB STORAGE】
1. Cov ntshav kuaj ntshav thiab ntshav kuaj tau sau rau hauv cov hlab uas muaj K2-EDTA tuaj yeem siv rau cov khoom siv no.
2. Cov khoom yuav tsum tau capped thiab yuav khaws cia ntev txog 48 teev ntawm 2 ° C - 8 ° C ua ntej kev soj ntsuam.
Cov khoom khaws cia ntev dua (txog 6 lub hlis) yuav tsum tau khov ib zaug ntawm -20 ° C ua ntej kev soj ntsuam.
Tsis txhob rov qab khov-thaw cycles.
PROTOCOL
【Kev npaj Reagent】
1. Tag nrho cov reagents yuav tsum tau muab tshem tawm ntawm lub tub yees thiab tso cai rov qab mus rau chav tsev kub ua ntej siv
(20 ° txog 25 ° C).Txuag tag nrho cov reagents hauv lub tub yees tam sim tom qab siv.
2. Tag nrho cov qauv thiab kev tswj yuav tsum tau vortexed ua ntej siv.
3. hACE2-HRP Solution Preparation: Dilute hACE2-HRP concentrate ntawm 1: 51 dilution ratio nrog Dilution
Tsis pub.Piv txwv li, dilute 100 μL ntawm hACE2-HRP mloog zoo nrog 5.0mL ntawm HRP Dilution Buffer rau
ua cov tshuaj hACE2-HRP.
4. 1 × Kev Npaj Kev Npaj: Dilute 20 × Ntxuav Tshuaj nrog deionized los yog distilled dej nrog ib tug
ntim piv ntawm 1:19.Piv txwv li, dilute 20 mL ntawm 20 × Ntxuav Solution nrog 380 mL ntawm deionized los yog
dej distilled los ua 400 mL ntawm 1 × Ntxuav Solution.
【Cov txheej txheem xeem】
1. Hauv cov raj cais, aliquot 120μL ntawm cov tshuaj npaj hACE2-HRP.
2. Ntxiv 6 μL ntawm calibrators, tsis paub cov qauv, kev tswj zoo hauv txhua lub raj thiab sib tov zoo.
3. Hloov 100μL ntawm txhua qhov sib tov npaj rau hauv kauj ruam 2 mus rau hauv cov dej sib txuas microplate raws li
rau predesigned xeem configuration.
3. Npog lub phaj nrog Plate Sealer thiab incubate ntawm 37 ° C rau 30 feeb.
4. Tshem lub Phaj Sealer thiab ntxuav lub phaj nrog kwv yees li 300 μL ntawm 1 × Ntxuav Tshuaj rau plaub zaug.
5. Coj mus rhaub lub phaj ntawm daim ntawv phuam kom tshem tawm cov kua seem hauv qhov dej tom qab ntxuav cov kauj ruam.
6. Ntxiv 100 μL ntawm TMB Tshuaj rau txhua qhov dej thiab ua rau lub phaj hauv qhov tsaus ntuj ntawm 20 - 25 ° C rau 20 feeb.
7. Ntxiv 50 μL ntawm Stop Solution rau txhua qhov dej kom tsis txhob muaj cov tshuaj tiv thaiv.
8. Nyeem cov absorbance hauv microplate nyeem ntawv ntawm 450 nm hauv 10 feeb (630nm raws li cov khoom siv yog
pom zoo rau kev ua tau zoo dua precision).
