SARS-CoV-2 Neutralizing Antibody Detection Kit (ELISA)
【PRINCIPLE】
Iyo SARS-CoV-2 Neutralizing Antibody Detection Kit yakavakirwa pamakwikwi eELISA maitiro.
Kushandisa purified receptor binding domain (RBD), protein kubva kune viral spike (S) protein uye iyo host cell.
receptor ACE2, iyi bvunzo yakagadzirirwa kutevedzera hutachiona-host neutralizing kupindirana.
Calibrator, Hunhu Kudzora, uye serum kana plasma samples inosanganiswa yega zvakanaka mukunatswa
buffer ine haACE2-HRP conjugate yakatorwa mumachubhu madiki. Zvadaro masanganiswa anoendeswa mukati
iyo microplate matsime ane immobilized recombinant SARS-CoV-2 RBD fragment (RBD) ye
incubation. Munguva yemakumi matatu-maminetsi incubation, iyo RBD yakananga antibody mune calibrators, QC uye
masampuli anozokwikwidza nehACE2-HRP kune chaiyo inosunga iyo RBD isingafambe mumatsime. Mushure
iyo incubation, matsime anoshambidzwa ka4 kuti abvise unbound haACE2-HRP conjugate. Mhinduro ye
TMB inozowedzerwa yoiswa maminetsi makumi maviri patembiricha yemumba, zvichikonzera kugadzirwa kwe
ruvara rwebhuruu. Iko kukura kwemavara kunomiswa nekuwedzera kwe1N HCl, uye kunyura ndiko
kuyerwa spectrophotometrically pa450 nm. Kusimba kwemuvara wakaumbwa kunoenderana ne
huwandu hwe enzyme iripo, uye inopesana nehuwandu hwemazinga akaedzwa nenzira imwecheteyo.
Kuburikidza nekuenzanisa necalibration curve inoumbwa neakapihwa macalibrator, iyo concentration ye
kutapudza masoja ekudzivirira chirwere mumuenzaniso usingazivikanwi anobva averengwa.
【ZVINHU ZVINODIWA ASI ZVISINA KUPIWA】
1. Mvura yakasvibiswa kana yakasvibiswa
2. Mapaipi akanyatsojeka: 10μL, 100μL, 200μL uye 1 mL
3. Kuraswa pipette mazano
4. Microplate reader inokwanisa kuverenga kunyura pa450nm.
5. Bepa rinopinza
6. Pepa regirafu
7. Vortex mixer kana yakaenzana
【SPECIMN KUCHENGA UYE KUCHENGETA】
1. Serum nePlasma samples dzakaunganidzwa mumachubhu ane K2-EDTA anogona kushandiswa pakiti iyi.
2. Mienzaniso inofanira kuvharwa uye inogona kuchengetwa kusvika kumaawa 48 pa 2 °C - 8 °C isati yaedzwa.
Mienzaniso inochengetwa kwenguva yakareba (kusvika kumwedzi mitanhatu) inofanira kuomeswa kamwe chete pa -20 °C isati yaedzwa.
Dzivisa kudzokororwa kwechando-kunyungudika kutenderera.
PROTOCOL
【Reagent Kugadzirira】
1. Mareagents ese anofanirwa kubviswa mufiriji uye kubvumidzwa kudzokera kune tembiricha yekamuri isati yashandiswa
(20° kusvika 25°C). Sevha ese mareagents mufiriji nekukurumidza mushure mekushandisa.
2. Yese sampuli uye zvidzoreso zvinofanirwa kuvharwa zvisati zvashandiswa.
3. haACE2-HRP Kugadzirira Kugadzirira: Dilute haACE2-HRP concentrate pa1: 51 dilution ratio neDilution
Buffer. Semuyenzaniso, dilute 100 μL yehACE2-HRP inonangana ne5.0mL yeHRP Dilution Buffer kusvika.
gadzira mhinduro yehACE2-HRP.
4. 1× Wash Solution Kugadzirira: Dilute the 20× Wash Solution nemvura yakasvibiswa kana yakasvibiswa ne
vhoriyamu reshiyo ye1:19. Semuyenzaniso, dilute 20 mL ye 20× Geza Solution ne 380 mL ye deionized kana
mvura yakasvibiswa kuita 400 mL ye1 × Wash Solution.
【Test Procedure】
1. Mumachubhu akasiyana, aliquot 120μL yehACE2-HRP Solution yakagadzirwa.
2. Wedzera 6 μL ye calibrator, sampuli dzisingazivikanwi, maitiro ehutano muhubhu imwe neimwe uye sanganisa zvakanaka.
3. Kuendesa 100μL yemusanganiswa wega wega wakagadzirirwa mudanho rechipiri mumatsime emicroplate anoenderana.
kune predesigned test configuration.
3. Vhara ndiro yacho nePlate Sealer uye isa pa 37°C kwemaminitsi makumi matatu.
4. Bvisa Plate Sealer wogeza ndiro ne300 μL ye 1× Geza Solution patsime kane.
5. Bata ndiro pajira repapepa kuti ubvise mvura yakasara mumatsime mushure mekugeza matanho.
6. Wedzera 100 μL yeTMB Solution patsime rega rega uye isa ndiro murima pa20 - 25 ° C kwemaminitsi makumi maviri.
7. Wedzera 50 μL yeStop Solution patsime rega rega kuti umise maitiro.
8. Verenga kunyura mune microplate kuverenga pa450 nm mukati megumi maminetsi (630nm sechinhu chinowedzera.
inokurudzirwa kuita kwepamusoro chaiko).