Ob-stranded RNA (dsRNA) ELISA KIT

Cov khoom siv no yog Enzyme-Linked Immunosorbent Assay (ELISA) coupling nrog biotin-Streptavidin system, rau kev ntsuas ntau ntawm dsRNA nrog ntev tshaj 60 lub hauv paus khub (bp), tsis hais seb yuav ua li cas.Lub phaj tau ua ntej coated nrog anti-dsRNA antibody.dsRNA tam sim no nyob rau hauv cov qauv yog ntxiv thiab khi rau cov tshuaj tiv thaiv kab mob coated rau ntawm qhov dej.Thiab tom qab ntawd biotinylated anti-dsRNA antibody ntxiv thiab khi rau dsRNA hauv cov qauv.Tom qab ntxuav, HRP-Streptavidin ntxiv thiab khi rau Biotinylated anti-dsRNA antibody.Tom qab incubation unbound HRP-Streptavidin yog ntxuav tawm.Tom qab ntawd TMB substrate tov yog ntxiv thiab catalyzed los ntawm HRP los tsim cov khoom xim xiav uas hloov mus rau daj tom qab ntxiv acidic nres.Qhov ceev ntawm daj yog proportional rau lub hom phiaj tus nqi ntawm dsRNA ntes nyob rau hauv phaj.Lub absorbance yog ntsuas ntawm 450 nm.

Daim ntawv thov

Cov khoom siv no yog rau kev ntsuas ntau ntawm dsRNA residual.

Cov khoom siv

Cia thiab ruaj khov

1. Rau cov khoom siv tsis siv: Tag nrho cov khoom siv tuaj yeem khaws cia ntawm 2 ~ 8 ℃ hauv txee lub neej.Lub teeb muaj zog yuav tsum zam kom ruaj khov.

2. Rau cov khoom siv siv: Thaum lub microplate qhib, thov npog cov qhov dej tsis siv nrog lub phaj sealer thiab rov qab mus rau lub hnab ntawv uas muaj cov khoom siv desiccant, zip-seal lub hnab ntawv ci thiab rov qab mus rau 2 ~ 8 ℃ sai li sai tau tom qab siv.Lwm cov reagents yuav tsum tau rov qab mus rau 2 ~ 8 ℃ sai li sai tau tom qab siv.

Cov ntaub ntawv xav tau tab sis tsis muab

1. Microplate nyeem ntawv nrog 450 ± 10nm lim (zoo dua yog tias tuaj yeem ntes ntawm 450 thiab 650 nm wavelength).

2. Microplate shaker.

3. RNase-dawb cov lus qhia thiab centrifuge raj.

Kev Ua Haujlwm Flowchart

Ua ntej Koj Pib

1. Nqa tag nrho cov khoom siv thiab cov qauv mus rau chav sov (18-25 ℃) ua ntej siv.Yog tias cov khoom siv yuav tsis siv nyob rau hauv ib zaug, thov tsuas yog tshem tawm cov strips thiab reagents rau qhov kev sim tam sim no, thiab tso cov strips thiab reagents nyob rau hauv qhov xav tau.

2. Ntxuav buffer: dilute 40mL ntawm 20 × concentrated ntxuav buffer nrog 760mL ntawm deionized los yog distilled dej los npaj 800mL ntawm 1 × ntxuav buffer.

3. Txuj: luv luv tig los yog centrifuge cov khoom lag luam ua ntej siv.Qhov concentration ntawm plaub tus qauv muab yog 5ng / μL.Rau UTP thiab pUTP dsRNA cov qauv, thov dilute cov Tshuag tov rau 1,0.5,0.25,0.125,0.0625,0.0312,0.0156,0pg/μL nrog STE tsis rau kos tus qauv nkhaus.Rau N1-Me-pUTP dsRNA cov qauv, thov dilute cov tshuaj Tshuag rau 2,1,0.5,0.25,0.125,0.0625,0.0312, 0pg/μL nrog STE tsis tuaj yeem kos tus qauv nkhaus.Rau 5-OMe-UTP dsRNA tus qauv, thov dilute cov tshuaj Tshuag rau 4,2,1,0.5, 0.25,0.125,0.0625, 0pg / μL nrog STE tsis tuaj yeem kos tus qauv nkhaus.Peb pom zoo cov qauv tuaj yeem diluted raws li cov kab kos hauv qab no:

4. Biotinylated detection antibody thiab HRP-streptavidin ua hauj lwm tov: luv luv spin los yog centrifuge cov Tshuag tov ua ntej siv.Dilute lawv mus rau qhov ua hauj lwm concentration nrog dilution tsis.

5. TMB substate: aspirate qhov xav tau ntau npaum li cas ntawm cov tshuaj nrog sterilized tswv yim thiab tsis txhob muab cov residual tov rau hauv lub vial dua.TMB substate yog rhiab rau lub teeb, tsis txhob raug TMB substrate rau lub teeb ntev.

Siv raws tu qauv

1. Txiav txim seb tus naj npawb ntawm cov strips xav tau rau qhov kev xeem.Ntxig cov strips rau hauv cov thav ntawv rau kev siv.Cov phaj seem uas tsis tau siv rau hauv qhov kev ntsuam xyuas no yuav tsum tau muab ntim rau hauv lub hnab nrog desiccant.Kaw lub hnab kom nruj rau lub tub yees cia.

2. Ntxiv 100μL txhua qhov dilutions ntawm tus qauv, dawb paug thiab cov qauv mus rau hauv cov qhov dej tsim nyog.Npog nrog lub phaj sealer.Incubate rau 1hr ntawm chav tsev kub nrog co ntawm 500rpm.Cov qauv yuav tsum tau diluted nrog STE tsis rau kom tsim nyog concentration rau qhov tseeb.

3. Ntxuav cov kauj ruam: Aspirate cov tshuaj thiab ntxuav nrog 250μL ntxuav buffer rau txhua qhov dej thiab cia nws sawv ntsug rau 30s.Muab pov tseg tsis huv tag nrho los ntawm snapping lub phaj rau ntawm daim ntawv absorbent.Tag nrho ntxuav 4 zaug.

4. Ntxiv 100μL ntawm biotinylated nrhiav kom pom cov tshuaj tiv thaiv ua haujlwm rau hauv txhua qhov dej.Npog nrog lub phaj sealer.Incubate rau 1hr ntawm chav tsev kub nrog co ntawm 500rpm.

5. Rov ua kauj ruam ntxuav.

6. Ntxiv 100μL ntawm HRP-streptavidin ua haujlwm daws rau hauv txhua qhov dej.Npog nrog lub phaj sealer.Incubate rau 30min ntawm chav tsev kub nrog co ntawm 500rpm.

7. Rov ua cov kauj ruam ntxuav dua.

8. Ntxiv 100μL ntawm TMB substrate tov rau hauv txhua qhov dej.Npog nrog lub phaj sealer.Incubate rau 30 feeb ntawm RT Tiv thaiv los ntawm lub teeb.Cov kua yuav tig xiav los ntawm qhov sib ntxiv ntawm cov tshuaj substrate.

9. Ntxiv 50μL ntawm cov tshuaj nres rau hauv txhua qhov dej.Cov kua yuav tig daj los ntawm qhov sib ntxiv ntawm cov tshuaj nres.Tom qab ntawd khiav lub microplate nyeem ntawv thiab ntsuas qhov ntsuas ntawm 450nm tam sim ntawd.

Kev suav cov txiaj ntsig

1. Qhov nruab nrab qhov sib npaug ntawm kev nyeem ntawv rau txhua tus qauv, tswj, thiab cov qauv thiab rho tawm qhov nruab nrab xoom tus qauv kho qhov muag ntom ntom.Tsim tus qauv nkhaus nrog absorbance ntawm ntsug (Y) axis thiab dsRNA concentration ntawm kab rov tav (X) axis.

2. Nws raug nquahu kom ua qhov kev suav nrog lub khoos phis tawj-raws li nkhaus-fitting software xws li tus kws tshaj lij nkhaus 1.3 lossis ELISA Calc hauv 5 lossis 4 qhov ntsuas tsis yog-linear haum qauv.

Kev ua tau zoo

1. Sensitivity:

Kev kuaj pom qis dua: ≤ 0.001pg/μL (rau UTP-, pUTP-, N1-Me-pUTP-dsRNA), ≤ 0.01pg/μL (rau 5-OMe-UTP-dsRNA).

Kev txwv qis dua: 0.0156 pg/μL (rau UTP-, pUTP-dsRNA), 0.0312 pg/μL (rau N1-Me-pUTP-dsRNA), 0.0625 pg/μL (rau 5-OMe-UTP-dsRNA).

2. Precision: CV ntawm Intra-Assay ≤10%, CV ntawm Inter-Assay ≤10%

3. Rov qab: 80% ~ 120%

4.Linearity: 0.0156-0.5pg/μL(forUTP-,pUTP-dsRNA)0.0312-1pg/μL(forN1-Me-pUTP dsRNA), 0.0625-1pg/μL(rau 5-OMe-UTP-dsRNA).

Kev txiav txim siab

1. TMB cov tshuaj tiv thaiv kub thiab lub sijhawm yog qhov tseem ceeb, thov tswj lawv raws li cov lus qhia nruj me ntsis.

2. Txhawm rau kom ua tiav qhov kev soj ntsuam zoo rov tsim dua tshiab thiab rhiab heev, kev ntxuav kom zoo ntawm daim hlau kom tshem tawm cov tshuaj reacted ntau dhau yog qhov tseem ceeb.

3. Tag nrho cov reagents yuav tsum tau tov kom huv si ua ntej siv thiab tsis txhob bumbles thaum lub sij hawm ua qauv los yog reagents ntxiv.

4. Yog hais tias muaju tau tsim nyob rau hauv cov concentrated ntxuav tsis (20x), sov rau 37 ℃ thiab sib tov maj mam kom txog rau thaum cov muaju yog yaj tag.

5. Tsis txhob soj ntsuam cov qauv uas muaj Sodium Azide (NaN3), vim tias nws tuaj yeem rhuav tshem cov haujlwm HRP uas ua rau qis qis ntawm cov nyiaj dsRNA.

6. Zam tsis txhob muaj kab mob RNase thaum kuaj.

7. Tus qauv / qauv, kuaj pom cov tshuaj tiv thaiv kab mob thiab SA-HRP kuj tuaj yeem ua ntawm RT yam tsis muaj kev co, tab sis qhov no yuav ua rau kev tshawb pom rhiab heev los ntawm ib zaug.Rau qhov no, peb xav kom UTP thiab pUTP dsRNA cov qauv yuav tsum tau diluted los ntawm 2pg / μL, N1-Me-pUTP dsRNA cov qauv yuav tsum tau diluted los ntawm 4pg / μL thiab 5-OMe-UTP dsRNA tus qauv yuav tsum tau diluted los ntawm 8pg / μL.Tsis tas li ntawd, incubate HRP-streptavidin ua hauj lwm tov rau 60min ntawm chav tsev kub.Tsis txhob siv lub khob shaker, vim hais tias lub khob shaker yuav ua rau qhov tshwm sim tsis raug.

Yam tshwm sim

1. Cov ntaub ntawv nkhaus txheem

2. Txheem nkhaus xam

3. Liner nrhiav tau ntau yam: 0.0312- 1pg/μL