Ngwa SARS-CoV-2 na-anọpụ iche na-achọpụta ihe mgbochi (ELISA)
【Ụkpụrụ】
Ngwa SARS-CoV-2 Neutralizing Antibody Detection Kit dabere na usoro ELISA asọmpi.
Iji ngalaba nnabata nnabata dị ọcha (RBD), protein sitere na protein spike (S) na cell host
ACE2 nnabata, a na-eme nnwale a ka ọ dị ka mmekọrịta na-egbochi nje virus na-anabata ya.
A na-agwakọta calibrators, njikwa ogo, yana ihe nlele serum ma ọ bụ plasma n'otu n'otu na mgbaze
ihe nchekwa nwere hACE2-HRP conjugate akọrọ n'ime obere tubes. Mgbe ahụ, a na-ebufe ngwakọta ndị ahụ
olulu mmiri microplate nwere iberibe recombinant SARS-CoV-2 RBD (RBD) maka
incubation. N'ime nkeji iri atọ ahụ, ihe mgbochi RBD akọwapụtara na calibrators, QC na
samples ga-asọmpi na hACE2-HRP maka njide kpọmkwem RBD enweghị ike ime n'olulu mmiri. Mgbe
Mkpokọta ahụ, a na-asa olulu mmiri ugboro 4 iji wepụ conjugate hACE2-HRP na-adịghị. A ngwọta nke
A na-agbakwunye TMB ma tinye ya maka nkeji 20 n'ime ụlọ okpomọkụ, na-ebute mmepe nke a
agba anụnụ anụnụ. A na-akwụsị mmepe nke agba na mgbakwunye nke 1N HCl, na ntinye bụ
tụrụ spectrophotometrically na 450 nm. Ike nke agba kpụrụ bụ nhata na
ọnụọgụ nke enzyme dị ugbu a, ma na-ejikọta ya na ọnụ ọgụgụ nke ụkpụrụ enyochala n'otu ụzọ ahụ.
Site n'iji ya tụnyere usoro nhazi nke kpụrụ site na calibrators nyere, ịta nke
A na-agbakọ ọgwụ mgbochi mgbochi na-adịghị ahụkebe na nlele amabeghị.
【AKWỤKWỌ KWESỊRỊ MA Ọ BỤGHỊ】
1. Mmiri distilled ma ọ bụ deionized
2. pipettes nkenke: 10μL, 100μL, 200μL na 1 ml
3. Atụmatụ pipette na-atụfu
4. Microplate agụ ike ịgụ absorbance na 450nm.
5. Akwụkwọ absorbent
6. Akwụkwọ eserese
7. Vortex mixer ma ọ bụ dakọtara
【Mkpokọta na ichekwa ihe atụ】
1. Serum na Plasma samples anakọtara na tubes nwere K2-EDTA nwere ike iji maka ngwa a.
2. Ekwesịrị ịdebe ihe nlele ma chekwaa ya ruo awa 48 na 2 Celsius C - 8 Celsius C tupu e nyochaa ya.
Ụdị echere ogologo oge (ruo ọnwa 6) kwesịrị ka oyi kpọnwụrụ naanị otu ugboro na -20 Celsius C tupu nyocha.
Zenarị okirikiri ifriizi ugboro ugboro.
PROTOCOL
【Nkwadebe Reagent】
1. A ga-ewepụrịrị reagents niile na refrigeration wee kwe ka ha laghachi n'ime ụlọ okpomọkụ tupu ejiri ya
(20 ° C ruo 25 Celsius C). Chekwaa reagents niile na friji ozugbo ejirichara ya.
2. All samples na njikwa kwesịrị vortexed tupu ojiji.
3. hACE2-HRP Nkwadebe Ngwọta: Dilute hACE2-HRP itinye uche na 1: 51 dilution ratio na Dilution
Ihe nchekwa. Dịka ọmụmaatụ, gbanye 100 μL nke hACE2-HRP uche na 5.0mL nke HRP Dilution Buffer ka
mee ihe ngwọta hACE2-HRP.
4. Nkwadebe Ngwọta Ngwọta 1 ×: Jiri mmiri deionized ma ọ bụ distilled gbanye ihe ngwọta 20 ×.
Oke oke nke 1:19. Dịka ọmụmaatụ, gbanye 20 ml nke 20 × Wash Solution na 380 ml nke deionized ma ọ bụ
mmiri distilled iji mee 400 ml nke 1 × Ngwọta Ngwọta.
【Usoro ule】
1. Na tubes dị iche iche, aliquot 120μL nke ngwọta hACE2-HRP a kwadebere.
2. Tinye 6 μL nke calibrators, ihe ndị a na-amaghị, njikwa mma na tube ọ bụla ma jikọta nke ọma.
3. Nyefee 100μL nke ngwakọta ọ bụla a kwadebere na nzọụkwụ 2 n'ime olulu mmiri microplate kwekọrọ.
na nhazi ule eburu ụzọ chepụta.
3. Na-ekpuchi efere ahụ na Plate Sealer ma tinye ya na 37 ° C maka nkeji 30.
4. Wepu ihe mkpuchi efere ma sachaa efere ahụ na ihe dịka 300 μL nke 1 × Wash Solution kwa olulu mmiri ugboro anọ.
5. Kpatụ efere na akwa nhicha akwụkwọ iji wepụ mmiri mmiri fọdụrụ na olulu mmiri mgbe ịsachara nzọụkwụ.
6. Tinye 100 μL nke TMB Ngwọta na nke ọ bụla ma tinye efere ahụ n'ọchịchịrị na 20 - 25 ° C maka nkeji 20.
7. Tinye 50 μL nke Ngwọta Kwụsị na olulu mmiri ọ bụla iji kwụsị mmeghachi omume.
8. Gụọ absorbance na microplate reader na 450 nm n'ime nkeji 10 (630nm dị ka ngwa.
akwadoro maka ịrụ ọrụ nkenke dị elu).