SARS-CoV-2 Neutralizing Antibody Detection Kit (ELISA)
【MFUNDO】
SARS-CoV-2 Neutralizing Antibody Detection Kit idakhazikitsidwa ndi njira yampikisano ya ELISA.
Pogwiritsa ntchito purified receptor binding domain (RBD), mapuloteni ochokera ku viral spike (S) protein ndi cell host.
receptor ACE2, mayesowa adapangidwa kuti atsanzire kuyanjana kwa ma virus-host neutralizing.
Ma Calibrators, Quality Controls, ndi seramu kapena plasma zitsanzo zimasakanizidwa bwino mu dilution
buffer yokhala ndi haACE2-HRP conjugate aliquote mu machubu ang'onoang'ono.Kenako zosakaniza zimasamutsidwa mkati
zitsime za microplate zomwe zili ndi chidutswa chosasunthika cha SARS-CoV-2 RBD (RBD) cha
kuyamwitsa.Pamakulitsidwe amphindi 30, ma antibody enieni a RBD mu ma calibrators, QC ndi
zitsanzo zidzapikisana ndi haACE2-HRP pomanga mwachindunji RBD yosasunthika m'zitsime.Pambuyo
poyikira, zitsime zimatsuka nthawi 4 kuti muchotse conjugate ya haACE2-HRP yosamangidwa.Yankho la
TMB imawonjezeredwa ndi kukulungidwa kwa mphindi 20 kutentha kwa firiji, zomwe zimapangitsa kuti a
mtundu wabuluu.Kukula kwamtundu kumayimitsidwa ndikuwonjezera kwa 1N HCl, ndipo kuyamwa ndi
kuyeza spectrophotometrically pa 450 nm.Kuchuluka kwa mtundu wopangidwa ndi wofanana ndi
kuchuluka kwa ma enzyme omwe alipo, ndipo amagwirizana mosagwirizana ndi kuchuluka kwa miyezo yoyesedwa mwanjira yomweyo.
Kupyolera mu kuyerekezera ndi ma calibration pamapindikira opangidwa ndi ma calibrator operekedwa, kuchuluka kwa
Kuchepetsa ma antibodies mu zitsanzo zosadziwika kumawerengedwa.
【ZINTHU ZOFUNIKA KOMA ZOSAPATSIDWA】
1. Madzi osungunuka kapena osungunuka
2. Mapaipi olondola: 10μL, 100μL, 200μL ndi 1mL
3. Malangizo a pipette otayika
4. Microplate reader amatha kuwerenga absorbance pa 450nm.
5. Pepala loyamwa
6. Mapepala a graph
7. Chosakaniza cha Vortex kapena chofanana
【KUSONGA ZINTHU NDIPONSO KUSUNGA】
1. Zitsanzo za seramu ndi Plasma zosonkhanitsidwa m'machubu okhala ndi K2-EDTA zingagwiritsidwe ntchito pa zidazi.
2. Zitsanzo ziyenera kutsekedwa ndipo zikhoza kusungidwa kwa maola 48 pa 2 °C - 8 °C musanayese.
Zitsanzo zomwe zimasungidwa kwa nthawi yayitali (mpaka miyezi 6) ziyenera kusungidwa kamodzi kokha pa -20 ° C musanayese.
Pewani kuzizira kobwerezabwereza.
PROTOCOL
【Kukonzekera kwa Reagent】
1. Ma reagents onse ayenera kuchotsedwa mufiriji ndikuloledwa kubwereranso kutentha kwa chipinda musanagwiritse ntchito
(20 ° mpaka 25 ° C).Sungani ma reagents onse mufiriji mukangogwiritsa ntchito.
2. Zitsanzo zonse ndi zowongolera ziyenera kutsekedwa musanagwiritse ntchito.
3. HACE2-HRP Yankho Kukonzekera: Dilute hACE2-HRP concentrate pa 1: 51 dilution ratio ndi Dilution
Bafa.Mwachitsanzo, chepetsani 100 μL ya haACE2-HRP yokhazikika ndi 5.0mL ya HRP Dilution Buffer mpaka
pangani yankho la haACE2-HRP.
4. 1 × Sambani Njira Yothetsera Kukonzekera: Sungunulani 20× Sambani Solution ndi madzi osungunuka kapena osungunuka ndi madzi.
kuchuluka kwa voliyumu 1:19.Mwachitsanzo, tsitsani 20 ml ya 20× Sambani Solution ndi 380 ml ya deionized kapena deionized.
madzi osungunuka kuti apange 400 mL ya 1 × Sambani Solution.
【Njira Yoyesera】
1. M'machubu osiyana, aliquot 120μL ya HACE2-HRP Solution yokonzedwa.
2. Onjezani 6 μL ya ma calibrator, zitsanzo zosadziwika, zowongolera zamtundu uliwonse mu chubu ndikusakaniza bwino.
3. Tumizani 100μL ya kusakaniza kulikonse kokonzedwa mu gawo 2 mu zitsime zofananira za microplate molingana
ku kasinthidwe koyeserera kokonzedweratu.
3. Phimbani mbale ndi Plate Sealer ndipo muyimire pa 37 ° C kwa mphindi 30.
4. Chotsani Chosindikizira mbale ndikutsuka mbaleyo ndi pafupifupi 300 μL ya 1× Sambani Solution pa chitsime kanayi.
5. Dinani mbaleyo papepala kuti muchotse madzi otsalira m'zitsime mutatsuka masitepe.
6. Onjezani 100 μL ya TMB Solution pachitsime chilichonse ndikuukira mbale mumdima wa 20 - 25 ° C kwa mphindi 20.
7. Onjezani 50 μL ya Stop Solution pachitsime chilichonse kuti muyimitse zomwe zikuchitika.
8. Werengani kuyamwa kwa microplate reader pa 450 nm mkati mwa mphindi 10 (630nm monga chowonjezera ndi
zolimbikitsidwa kuti zizichita bwino kwambiri).
