Sarrafa-CoV-2 Na'urar Gano Maganin Neutralizing Antibody (ELISA)
【KA'IDA】
Kit ɗin Ganewar Ganewa na Tsabtace Tsabtace Tsakanin SARS-CoV-2 ya dogara ne akan dabarar ELISA mai gasa.
Yin amfani da yanki mai ɗaurin mai karɓa mai tsafta (RBD), furotin daga furotin mai saurin kamuwa da cuta (S) da tantanin halitta
mai karɓar ACE2, an ƙirƙiri wannan gwajin don yin kwaikwayi ma'amala mai hana ƙwayoyin cuta.
Calibrators, Ingantattun Gudanarwa, da samfuran jini ko samfuran plasma ana gauraye su da kyau a cikin dilution
buffer mai dauke da hACE2-HRP conjugate aliquoted a cikin kananan bututu. Sa'an nan kuma gauraye suna canjawa wuri a ciki
rijiyoyin microplate da ke dauke da recombinant recombinant SARS-CoV-2 RBD guntu (RBD) don
shiryawa. A lokacin shiryawa na mintuna 30, takamaiman rigakafin RBD a cikin calibrators, QC da
samfurori za su yi gasa tare da hACE2-HRP don takamaiman ɗaure RBD da ba a iya motsi a cikin rijiyoyin. Bayan
da shiryawa, ana wanke rijiyoyin sau 4 don cire haɗin hACE2-HRP wanda ba a ɗaure ba. Magani na
Ana ƙara TMB kuma a sanya shi na tsawon minti 20 a cikin dakin da zafin jiki, yana haifar da ci gaban a
launin shuɗi. An dakatar da ci gaban launi tare da ƙari na 1N HCl, kuma abin sha shine
spectrophotometrically a 450 nm. Ƙarfin launi da aka kafa yana daidai da
adadin enzyme da ke nan, kuma yana da alaƙa da alaƙa da adadin ma'aunin da aka tantance ta hanya ɗaya.
Ta hanyar kwatanta tare da ma'auni na daidaitawa da aka samar ta hanyar calibrators da aka bayar, ƙaddamarwa na
neutralizing antibodies a cikin da ba a sani ba samfurin sai a lissafta.
【KAYAN DA AKE BUKATA AMMA BA'A SAMU BA】
1. Ruwan da aka distilled ko datti
2. Madaidaicin pipettes: 10μL, 100μL, 200μL da 1 ml
3. Tukwici pipette da za a iya zubarwa
4. Microplate reader iya karanta absorbance a 450nm.
5. Takarda mai sha
6. Takardar hoto
7. Vortex mahautsini ko daidai
【TATTAUNAWA DA MISALIN】
1. Ana iya amfani da samfuran jini da Plasma da aka tattara a cikin bututu masu ɗauke da K2-EDTA don wannan kayan.
2. Ya kamata a rufe samfurori kuma ana iya adana su har zuwa sa'o'i 48 a 2 ° C - 8 ° C kafin a tantance.
Samfuran da aka riƙe na tsawon lokaci (har zuwa watanni 6) yakamata a daskare su sau ɗaya kawai a -20 ° C kafin a tantance.
Guji maimaita daskarewa-narkewa.
PROTOCOL
【Shiri Reagent】
1. Dole ne a fitar da duk reagents daga firiji kuma a bar su su koma cikin zafin jiki kafin amfani
(20 ° zuwa 25 ° C). Ajiye duk reagents a cikin firiji da sauri bayan amfani.
2. Duk samfurori da sarrafawa ya kamata a yi vortexed kafin amfani.
3. HACE2-HRP Magani Shiri: Tsarma hACE2-HRP maida hankali a 1: 51 dilution rabo tare da Dilution
Buffer. Misali, tsarma 100 μL na hACE2-HRP maida hankali tare da 5.0ml na HRP Dilution Buffer zuwa
Yi maganin hACE2-HRP.
4. 1× Wash Magani Shiri: Tsarma 20× Wash Magani da deionized ko distilled ruwa tare da
rabon girma na 1:19. Misali, tsoma 20 ml na Maganin Wanke 20×20 tare da 380 ml na deionized ko
distilled ruwa don yin 400 ml na 1 × Wash Magani.
【Tsarin Gwaji】
1. A cikin tubes daban-daban, aliquot 120μL na maganin hACE2-HRP da aka shirya.
2. Ƙara 6 μL na calibrators, samfurori da ba a sani ba, masu sarrafawa masu inganci a cikin kowane bututu da haɗuwa da kyau.
3. Canja wurin 100μL na kowane cakuda da aka shirya a cikin mataki na 2 a cikin rijiyoyin microplate daidai.
zuwa tsarin gwajin da aka riga aka tsara.
3. Rufe farantin tare da Plate Seler kuma a sanya shi a 37 ° C na minti 30.
4. Cire Plate Seler kuma a wanke farantin tare da kusan 300 μL na 1 × Wash Solution kowace rijiya har sau hudu.
5. Matsa farantin a kan tawul ɗin takarda don cire ragowar ruwa a cikin rijiyoyin bayan matakan wankewa.
6. Ƙara 100 μL na Maganin TMB zuwa kowace rijiya kuma a saka farantin cikin duhu a 20 - 25 ° C na minti 20.
7. Ƙara 50 μL na Maganin Tsaya zuwa kowace rijiya don dakatar da amsawa.
8. Karanta absorbance a cikin microplate reader a 450 nm a cikin minti 10 (630nm kamar yadda m.
an ba da shawarar don mafi girman aiki daidai).