Competitive Enzyme Immunoassay Kit for Quantitative Analysis of Tylosin
Competitive Enzyme Immunoassay Kit rau
Quantitative Analysis ntawmTylosin
1. Keeb kwm
Tylosinyog tshuaj tua kab mob macrolide, uas yog siv los ua tshuaj tua kab mob thiab tiv thaiv mycoplasma.Kev nruj MRLs tau tsim vim tias cov tshuaj no tuaj yeem ua rau muaj kev phiv loj hauv qee pawg.
Cov khoom siv no yog cov khoom siv tshiab raws li ELISA thev naus laus zis, uas yog ceev, yooj yim, raug thiab rhiab piv nrog cov cuab yeej ntsuas thiab tsuas yog xav tau 1.5 teev hauv ib qho kev ua haujlwm, nws tuaj yeem txo qis kev ua haujlwm yuam kev thiab ua haujlwm hnyav.
2. Test Txoj Cai
Cov khoom siv no yog raws li kev sib tw tsis ncaj-kev sib tw ELISA thev naus laus zis.Lub qhov dej microtiter yog coated nrog coupling antigen.Tylosin residue hauv cov qauv sib tw nrog cov antigen coated ntawm microtiter phaj rau cov tshuaj tiv thaiv.Tom qab qhov sib ntxiv ntawm enzyme sau npe tshuaj tiv thaiv kab mob, TMB substrate yog siv los qhia cov xim.Kev nqus ntawm cov qauv yog qhov tsis zoo cuam tshuam rau tylosin nyob hauv nws, tom qab muab piv nrog Standard Curve, muab faib los ntawm cov khoom siv dilution, tylosin residue kom muaj nuj nqis hauv cov qauv tuaj yeem xam.
3. Cov ntawv thov
Cov khoom siv no tuaj yeem siv tau rau hauv kev txheeb xyuas qhov ntau thiab zoo ntawm tylosin residue hauv cov nqaij tsiaj (nqaij qaib, nqaij npuas, os) thiab mis nyuj, zib ntab, qe, thiab lwm yam.
4. Kev sib cuam tshuam
Tylosin……………………………………………………..100%
Tilmicosin…………………………………………………… <2%
5. Cov ntaub ntawv uas yuav tsum tau muaj
5.1 Cov khoom siv:
---- Microtiter phaj spectrophotometer (450nm / 630nm)
---- Rotary evaporator lossis nitrogen drying instruments
---- homogenizer
---- Shaker
---- Centrifuge
---- Kev ntsuas qhov sib npaug (inductance: 0.01g)
---- Kawm tiav pipette: 10ml
---- Roj hmab pipette qhov muag teev
---- Lub raj mis ntim: 10ml
---- Polystyrene centrifuge tubes: 50ml
---- Micropipettes: 20-200ml, 100-1000ml
250 ml - ntau yam
5.2 Reagents:
----Sodium hydroxide (NaOH, AR)
---- Sodium bicarbonate (NaHCO3,AR)
---- Sodium carbonate (NaCO3, AR)
---- Trichloroacetic acid (AR)
---- Acetonitrile (AR)
---- Ethyl acetate (AR)
┅┅n-Hexane (AR)
---- Deionized dej
6. Cov Khoom Siv Khoom Siv
l Microtiter phaj nrog 96 qhov dej coated nrog antigen
l Txuj kev daws teeb meem (5 fwj, 1ml / lub raj mis)
0 ppb, 0.5 ppb, 1.5 ppb, 4.5 ppb, 13.5 ppb
l Spiking txheem tswj: (1ml / lub raj mis)1ppm ua
l Enzyme conjugate 1ml ……………………………….
l Antibody tov 7ml……………………
l Solution A 7ml………………………………………
l Solution B 7ml ………………………………………. Red cap
l Nres tshuaj 7ml.………………………………….daj cap
l 20 × concentrated ntxuav kua 40ml
…………………………………………….. Transparent cap
l 4 × concentrated extraction tov 50ml
…………………………………………………….blue cap
7. Kev npaj Reagents:
Kev daws 1:0.1 mol/L NaOH tov
Qhov hnyav 0.4g NaOH rau 100ml deionized dej thiab sib tov tag nrho.
Kev daws 2: 1 mol/L NaOH tov
Qhov hnyav 4g NaOH rau 100ml deionized dej thiab sib tov tag nrho.
Kev daws 3: Carbonate tsis ntsev
Kev daws1: 0.2M PB
Dissolve 51,6 g ntawm Na2HPO4· 12 H2o,8,7g ua2PO4· 2 H2O nrog dej deionized thiab dilute rau 1000ml.
Kev daws2: Extraction tov
Dilute 2 × concentrated extraction tov nrog dej deionized nyob rau hauv lub ntim piv ntawm 1: 1 (Piv txwv li 10ml ntawm 2 × extraction tov + 10ml ntawm deionized dej), uas yuav siv tau rau cov qauv rho tawm,Cov tshuaj no tuaj yeem khaws cia ntawm 4 ℃ rau 1 lub hlis.
Kev daws3: Ntxuav kua
Dilute 20 × concentrated ntxuav tov nrog dej deionized nyob rau hauv lub ntim piv ntawm 1:19 (Piv txwv li 5ml ntawm 20 × ntxuav tov + 95ml ntawm deionized dej), uas yuav siv tau los ntxuav cov phiaj.Cov tshuaj no tuaj yeem khaws cia ntawm 4 ℃ rau 1 lub hlis.
8. Cov qauv npaj
8.1 Daim ntawv ceeb toom thiab ceev faj ua ntej ua haujlwm:
(a) Thov siv cov lus qhia ib zaug hauv cov txheej txheem ntawm kev sim, thiab hloov cov lus qhia thaum nqus cov reagent sib txawv.
(b) Nco ntsoov tias txhua yam cuab yeej huv si.
(c) Khaws cov ntaub so ntswg kom khov.
(d) Cov qauv npaj yuav tsum tau siv los ntsuas ib zaug.
8.2 Cov ntaub so ntswg (nqaij qaib, nqaij npuas, thiab lwm yam)
---- Homogenize tus qauv nrog homogenizer;
---- Siv 2.0 ± 0.05g ntawm homogenate rau hauv 50ml polystyrene centrifuge raj;ntxiv 2ml ntawm 0.2M PB (daws1) co kom yaj, thiab tom qab ntawd ntxiv 8ml ntawm ethyl acetate thiab co hnyav rau 3min;
---- Centrifuge rau kev sib cais: 3000g / ambient kub / 5min.
---- Hloov 4ml ntawm supernatant organic theem mus rau hauv 10ml iav raj, qhuav nrog 50-60 ℃ dej da dej hauv qab cov roj nitrogen;
---- Dissolve lub qhuav seem nrog 1ml ntawm n-hexane, vortex rau 30s kom yaj, thiab ces ntxiv 1ml ntawm extraction tov (daws2), vortex rau 1 min.centrifuge rau kev sib cais: 3000g / ambient kub / 5min
---- Tshem tawm lub supernatant n-hexane theem;noj 50μl ntawm lub substrate aqueous theem rau kev soj ntsuam.
Dilution yam: 1
8.2 tej
---- Siv 100μl ntawm cov qauv mis nyuj nyoos, sib tov nrog 900μl ntawm cov tshuaj extraction (daws2), thiab sib tov tag nrho.
---- Siv 50μl ntawm cov tshuaj npaj rau kev soj ntsuam.
Dilution yam: 10
9. Kev ntsuam xyuas
9.1 Daim ntawv ceeb toom ua ntej kev ntsuam xyuas
9.1.1 ibXyuas kom tag nrho cov reagents thiab microwells nyob rau hauv chav tsev kub (20-25 ℃).
9.1.2 ibRov qab tag nrho cov reagent seem rau 2-8℃tam sim ntawd tom qab siv.
9.1.3 ibNtxuav cov microwells kom raug yog ib kauj ruam tseem ceeb hauv kev ntsuam xyuas;Nws yog qhov tseem ceeb tshaj plaws rau kev rov ua dua tshiab ntawm ELISA tsom xam.
9.1.4a uavoid lub teeb thiab npog lub microwells thaum incubation.
9.2 Cov kauj ruam ntsuam xyuas
9.2.1 Nqa tag nrho cov reagents tawm ntawm chav tsev kub (20-25 ℃) rau ntau tshaj 30min, co maj mam ua ntej siv.
9.2.2 Tau txais cov microwells xav tau tawm thiab xa rov qab mus rau hauv lub hnab zip-lock ntawm 2-8 ℃ tam sim.
9.2.3 Lub diluted ntxuav kua yuav tsum tau rewarmed kom nyob rau hauv chav tsev kub ua ntej siv.
9.2.4 ibTus lej:Tus lej txhua txoj haujlwm microwell thiab tag nrho cov qauv thiab cov qauv yuav tsum tau khiav hauv qhov sib npaug.Sau cov qauv thiab cov qauv ua haujlwm.
9.2.5 ibAdd txheem tov / qauv thiab tshuaj tiv thaiv kab mob: Ntxiv 50µl ntawm tus qauv tov ((cov khoom muab)) los yog npaj qauv rau cov qhov dej sib thooj.Ntxiv 50µl ntawm cov tshuaj tiv thaiv kab mob (cov khoom muab).Sib tov maj mam los ntawm co lub phaj manually thiab incubate rau 30min ntawm 37 ℃ nrog npog.
9.2.6 ibNtxuav: Tshem lub hau npog maj mam thiab ntxuav cov kua dej tawm ntawm qhov dej thiab yaug cov microwells nrog 250µl diluted ntxuav kua (daws3) ntawm 10s rau 4-5 zaug.Nqus cov dej seem nrog cov ntawv nqus dej (qhov seem cua npuas tuaj yeem tshem tawm nrog cov lus tsis siv).
9.2.7 ibNtxiv enzyme conjugate: Ntxiv 100ml ntawm enzyme conjugate tov (cov khoom muab) rau txhua qhov dej, sib tov maj mam thiab incubate rau 30min ntawm 37 ℃ nrog npog.Rov ua kauj ruam ntxuav dua.
9.2.8 ibXim xim: Ntxiv 50µl ntawm kev daws A (cov khoom muab) thiab 50μl ntawm kev daws B (cov khoom muab) rau txhua qhov dej.Sib tov maj mam thiab incubate rau 15 feeb ntawm 37 ℃ nrog npog.
9.2.9 ibNtsuas: Ntxiv 50µl ntawm cov tshuaj nres (cov khoom muab) rau txhua qhov dej.Sib tov maj mam thiab ntsuas qhov nqus ntawm 450nm (Nws tau pom zoo ntsuas nrog dual-wavelength ntawm 450/630nm. Nyeem qhov tshwm sim hauv 5min tom qab ntxiv cov tshuaj nres).
10. Cov txiaj ntsig
10.1 Feem pua absorbance
Qhov nruab nrab qhov tseem ceeb ntawm absorbance qhov tseem ceeb tau txais rau cov qauv thiab cov qauv muab faib los ntawm tus nqi absorbance ntawm thawj tus qauv (zero standard) thiab muab faib ua 100%.Tus qauv xoom yog li ua kom sib npaug li 100% thiab cov txiaj ntsig absorbance tau hais hauv feem pua.
B
Absorbance (%) = —— × 100%
B0
B —— nqus tus qauv (los yog qauv)
B0 —— absorbance zero standard
10.2 Standard Curve
---- Txhawm rau kos tus qauv nkhaus: Siv tus nqi nqus ntawm cov qauv raws li y-axis, ib nrab logarithmic ntawm cov ntsiab lus ntawm tylosin qauv tov (ppb) raws li x-axis.
---- Lub tylosin concentration ntawm txhua tus qauv (ppb), uas tuaj yeem nyeem tau los ntawm qhov ntsuas qhov nkhaus, yog muab faib los ntawm qhov sib thooj Dilution yam ntawm txhua tus qauv ua raws, thiab qhov tseeb concentration ntawm cov qauv tau txais.
Thov ceeb toom:
software tshwj xeeb tau tsim los rau kev txheeb xyuas cov ntaub ntawv, uas tuaj yeem muab rau ntawm kev thov.
11. rhiab heev, raug thiab precision
Test Sensitivity:1.5p wb
Kev kuaj pom qhov txwv:
Tsiaj cov ntaub so ntswg……………………………………………………1.5ppb Mis ………………………………………………………………… 15 ppb Qhov tseeb:
Tsiaj cov ntaub so ntswg……………………………………………………80 ± 15%
Mis …………………………………………………………….80 ± 10%
Precision:
Variation coefficient ntawm ELISA cov khoom siv tsawg dua 10%.
12. Daim ntawv ceeb toom
12.1 Qhov nruab nrab qhov tseem ceeb ntawm qhov ntsuas qhov nqus tau txais rau cov qauv thiab cov qauv yuav raug txo yog tias cov reagents thiab cov qauv tsis tau raug tswj rau hauv chav tsev kub (20-25 ℃).
12.2 Tsis txhob cia cov microwells qhuav ntawm cov kauj ruam kom tsis txhob ua tsis tiav thiab ua haujlwm rau cov kauj ruam tom ntej tam sim ntawd tom qab coj mus rhaub lub microwells.
12.3 Co txhua reagent maj mam ua ntej siv.
12.4 Khaws koj cov tawv nqaij kom deb ntawm qhov kev daws teeb meem rau nws yog 0.5MH2SO4daws.
12.5 Tsis txhob siv cov khoom siv dhau los.Tsis txhob pauv cov reagents ntawm cov khoom sib txawv, lossis lwm yam nws yuav poob qhov rhiab heev.
12.6 Khaws cov khoom siv ELISA ntawm 2-8 ℃, tsis txhob khov.Seal so microwell daim hlau, Tsis txhob ncaj tshav ntuj thaum txhua qhov incubations.Npog cov phaj microtiter yog pom zoo.
12.7 Substrate tov yuav tsum tau tso tseg yog tias nws hloov xim.Cov reagents tuaj yeem hloov tsis zoo yog tias tus nqi nqus (450/630nm) ntawm tus qauv xoom tsawg dua 0.5 (A450nm <0.5).
12.8 Cov tshuaj tiv thaiv xim xav tau 15min tom qab ntxiv cov tshuaj A thiab tshuaj B. Thiab koj tuaj yeem ncua lub sijhawm incubation mus rau 20min lossis ntau dua yog tias cov xim teeb dhau los txiav txim siab.Tsis txhob tshaj 30min, ntawm qhov tsis sib xws, txo lub sij hawm incubation kom zoo.
12.9 Qhov zoo tshaj plaws tshuaj tiv thaiv kub yog 37 ℃.Kev kub siab los yog qis dua yuav ua rau muaj kev hloov pauv ntawm rhiab heev thiab absorbance qhov tseem ceeb.
13. Cia
Cia txias: 2-8 ℃.
Lub sijhawm cia: 12 lub hlis.
