I-SARS-CoV-2 Neutralizing Antibody Detection Kit (ELISA)
【ISIMISO】
I-SARS-CoV-2 Neutralizing Antibody Detection Kit isuselwe endleleni yokuncintisana ye-ELISA.
Kusetshenziswa isizinda esibophezelayo se-purified receptor (RBD), amaprotheni asuka ku-viral spike (S) protein kanye ne-host cell
i-receptor ACE2, lokhu kuhlola kuklanyelwe ukulingisa ukusebenzisana kwe-virus-host neutralizing.
Ama-Calibrator, Izilawuli Zekhwalithi, kanye namasampula e-serum noma e-plasma axutshwe ngawodwa kahle ekuhlanjululweni
ibhafa equkethe i-hACE2-HRP conjugate ecashunwe kumashubhu amancane. Khona-ke izingxube zidluliselwa phakathi
Imithombo ye-microplate equkethe i-immobilized recombinant SARS-CoV-2 RBD fragment (RBD)
ukufukamela. Ngesikhathi sokufukamela imizuzu engama-30, i-antibody ethize ye-RBD kuma-calibrator, i-QC kanye
amasampula azoqhudelana ne-hACE2-HRP ngokubopha ngokuqondile i-RBD enganyakazi emithonjeni. Ngemva
ekufukameleni, imithombo igezwa izikhathi ezi-4 ukuze kukhishwe i-hACE2-HRP conjugate engaboshiwe. Isixazululo se
I-TMB ibe seyengezwa futhi ifukamele imizuzu engu-20 ekamelweni lokushisa, okuholela ekwakhekeni kwe-a
umbala oluhlaza okwesibhakabhaka. Ukuthuthukiswa kombala kumisiwe ngokungezwa kwe-1N HCl, futhi ukumunca kuyinto
kukalwe i-spectrophotometrically ku-450 nm. Ukuqina kombala owenziwe kuhambisana ne
inani le-enzyme elikhona, futhi lihlobene ngokuphambene nenani lamazinga ahlolwe ngendlela efanayo.
Ngokuqhathanisa nejika lokulinganisa elakhiwe ama-calibrator anikeziwe, ukugxila kwe
amasosha omzimba anciphisa isampula engaziwa abe esebalwa.
【IZINTO EZIDINGEKAYO KODWA AKUNIKEZWE】
1. Amanzi acwengekile noma ahlanjululwe
2. Amapayipi anembile: 10μL, 100μL, 200μL no-1 mL
3. Amathiphu we-pipette alahlwayo
4. Umfundi we-Microplate okwazi ukufunda ukumunca ku-450nm.
5. Iphepha le-Absorbent
6. Iphepha legrafu
7. I-Vortex mixer noma okulinganayo
【UKUQOQWA KANYE NESIGCINISO】
1. Amasampula eSerum nePlasma aqoqwe kumashubhu aqukethe i-K2-EDTA angasetshenziswa kule kit.
2. Izibonelo kufanele zifakwe i-cap futhi zingagcinwa amahora angafika kwangu-48 ku-2 °C - 8 °C ngaphambi kokuhlolwa.
Izibonelo ezigcinwe isikhathi eside (kuze kufike ezinyangeni eziyisi-6) kufanele ziqandiswe kanye kuphela ku -20 °C ngaphambi kokuhlolwa.
Gwema imijikelezo yokuqhwaza ephindaphindiwe.
I-PROTOCOL
【Ukulungiswa kwe-Reagent】
1. Wonke ama-reagents kufanele akhishwe esiqandisini futhi avunyelwe ukuthi abuyele ekamelweni lokushisa ngaphambi kokusetshenziswa
(20° kuya ku-25°C). Gcina wonke ama-reagents esiqandisini ngokushesha ngemva kokusetshenziswa.
2. Wonke amasampuli nezilawuli kufanele zivuthwe ngaphambi kokusetshenziswa.
3. I-hACE2-HRP Ukulungiswa Kwesixazululo: Nciphisa i-hACE2-HRP ukugxilisa ingqondo ku-1: 51 dilution ratio ne-Dilution
Ibhafa. Isibonelo, hlanza u-100 μL we-hACE2-HRP ugxile ngo-5.0mL we-HRP Dilution Buffer ukuze
yenza isisombululo se-hACE2-HRP.
4. 1× Ukulungiselela Isixazululo Sokugeza: Nciphisa Isixazululo se-20× Wash ngamanzi akhiqiziwe noma acwecwe nge-
isilinganiso sevolumu 1:19. Isibonelo, hlambulula u-20 mL we-20× Wash Solution ngo-380 mL we-deionized noma
amanzi distilled ukwenza 400 mL of 1× Geza Isixazululo.
【Inqubo Yokuhlola】
1. Kumashubhu ahlukene, i-aliquot 120μL Yesixazululo se-hACE2-HRP esilungisiwe.
2. Engeza u-6 μL wezikali, amasampula angaziwa, izilawuli zekhwalithi kushubhu ngalinye futhi uhlanganise kahle.
3. Dlulisa u-100μL wengxube ngayinye elungiselelwe esinyathelweni sesi-2 emithonjeni ye-microplate ehambisanayo ngokuya
ekucushweni kokuhlola okuklanywe ngaphambili.
3. Mboza ipuleti nge-Plate Sealer bese ufukamela ku-37°C imizuzu engu-30.
4. Khipha I-Plate Sealer bese ugeza ipuleti cishe ngo-300 μL we-1× Geza Isixazululo somthombo izikhathi ezine.
5. Thinta ipuleti ithawula lephepha ukuze ukhiphe uketshezi olusele emithonjeni ngemva kokugeza izinyathelo.
6. Faka i-100 μL ye-TMB Isixazululo emthonjeni ngamunye bese ufukamela ipuleti ebumnyameni ngo-20 - 25°C imizuzu engu-20.
7. Engeza u-50 μL we-Stop Solution emthonjeni ngamunye ukuze umise ukusabela.
8. Funda ukumuncwa kumfundi we-microplate ku-450 nm phakathi nemizuzu eyi-10 (630nm njengesesekeli
kunconyelwe ukusebenza ngokunemba okuphezulu).