khoom

1. 1.Keeb kwm

Nitrofurans yog hluavtaws broad-spectrum tshuaj tua kab mob, uas nquag ua hauj lwm nyob rau hauv cov tsiaj tsim rau nws zoo heev antibacterial thiab pharmacokinetic zog.Lawv kuj tau siv los ua kev txhawb nqa kev loj hlob hauv npua, nqaij qaib thiab dej ntau lawm.Hauv cov kev tshawb fawb ntev nrog cov tsiaj txhu hauv lab tau qhia tias niam txiv cov tshuaj thiab lawv cov metabolites pom cov yam ntxwv carcinogenic thiab mutagenic.Qhov no tau ua rau muaj kev txwv tsis pub nitrofurans rau kev kho tsiaj uas siv rau kev tsim khoom noj.Cov tshuaj nitrofuran furaltadone, nitrofurantoin thiab nitrofurazone raug txwv tsis pub siv hauv cov khoom noj tsiaj hauv EU hauv xyoo 1993, thiab kev siv furazolidone raug txwv hauv xyoo 1995.

Kev tsom xam ntawm cov tshuaj nitrofuran residue yuav tsum tau ua raws li kev tshawb pom ntawm cov ntaub so ntswg khi metabolites ntawm nitrofuran niam txiv cov tshuaj.Txij li thaum niam txiv cov tshuaj tau metabolized sai heev, thiab cov ntaub so ntswg khi nitrofuran metabolites yuav khaws tau ntev, cov metabolites no tau siv los ua lub hom phiaj hauv kev tshawb nrhiav kev tsim txom ntawm nitrofurans, uas suav nrog Furazolidone metabolite (AOZ), Furaltadone metabolite (AMOZ. ), Nitrofurantoin metabolite (AHD) thiab Nitrofurazone metabolite (SEM).

AOZ-residues feem ntau txiav txim siab los ntawm LC-MS lossis LC-MS / MS.Enzyme immunoassays, piv nrog cov txheej txheem chromatographic, qhia qhov txiaj ntsig zoo ntawm kev nkag siab zoo, kev kuaj pom qhov txwv, cov cuab yeej siv thiab lub sijhawm xav tau.(Tus nqi sijhawm: 45 min)

1. 2.Test Principple

Cov khoom siv ELISA no yog tsim los kuaj AOZ raws li lub hauv paus ntsiab lus ntawm indirect-competitive enzyme immunoassay.Lub qhov dej microtiter yog coated nrog ntes BSA-txuas antigen.AOZ hauv cov qauv sib tw nrog cov antigen coated ntawm microtiter phaj rau cov tshuaj tiv thaiv ntxiv.Tom qab qhov sib ntxiv ntawm enzyme conjugate, chromogenic substrate yog siv thiab cov teeb liab yog ntsuas los ntawm spectrophotometer.Kev nqus yog inversely proportional rau AOZ concentration hauv cov qauv.

1. 3.Daim ntawv thov

Cov khoom siv no tuaj yeem siv rau hauv qhov ntau thiab qhov zoo ntawm AOZ residue hauvanima cov ntaub so ntswg(cov leeg, daim siab thiab lwm yam), zib mu.

1. 4.Kev sib tshuam

Furazolidone metabolite (AOZ) ………………..100%

Furaltadone metabolite (AMOZ) …………………… <0.1%

Nitrofurantoin metabolite (AHD) …………………… <0.1%

Nitrofurazone metabolite (SEM) ………………………………… <0.1%

Furazolidone …………………………………………………… 16.3%

Furaltadone …………………………………………………….<1%

Nitrofurantoin ……………………………………………..<1%

Nitrofurazone …………………………………………………….<1%

1. 5.Cov ntaub ntawv xav tau

5.1Cov khoom siv

---- Microtiter phaj spectrophotometer (450nm / 630nm)

---- Rotary evaporator lossis nitrogen drying instruments

---- Homogenizer / plab hnyuv

---- Shaker

---- Vortex tov khoom

---- Centrifuge

---- Kev ntsuas qhov sib npaug (inductance: 0.01g)

---- Kawm tiav pipette: 10ml

---- Roj hmab pipette qhov muag teev

---- Lub raj mis ntim: 100ml

---- Lub raj mis: 10ml

---- Polystyrene centrifuge raj: 2ml, 50ml

---- Micropipettes: 20ul-200ul, 100ul-1000ul,

250 ul-multipipette

5.2Reagents

---- Ethyl acetate (AR)

---- n-hexane (los yog n-heptane) (AR)

---- Dipotassium hydrogen phosphate trihydrate

(K₂HPO₄.3 H₂O) (AR)

---- Concentrated hydrochloric acid (HCl, AR)

----- Methanol

---- Sodium hydroxide (NaOH, AR)

---- Deionized dej

1. 6.Cov khoom siv

l Microtiter phaj coated nrog antigen, 96 qhov dej

l Txuj kev daws teeb meem (6 fwj, 1ml / lub raj mis)

0 ppb, 0.025 ppb, 0.075 ppb, 0.225 ppb, 0.675 ppb, 2.025 ppb

l Spiking txheem tswj: (1ml / lub raj mis) ......100 ppb

l Concentrated enzyme conjugate 1ml......liab cap

l Enzyme conjugate diluents 10ml …………..ntsuab cap

l Substrate A 7ml…………………………………………..…..

l Substrate B 7ml………………………………………………….…..

l Nres tshuaj 7ml……………………………………… daj cap

l 20 × concentrated ntxuav kua 40ml

………………………………………….……pob tshab cap

l 2 × concentrated extraction tov 60ml….blue cap

l 2-Nitrobenzaldehyde 15.1mg…………………

1. 7.Kev npaj Reagents

Kev daws 1Tom ntej: derivative reagent:

Ntxiv 10ml ntawm methanol rau lub raj mis uas muaj 2-Nitrobenzaldehyde thiab yaj.(ntawm qhov concentration ntawm 10mM).

Kev daws 2:0.1 kwm₂HPO₄tshuaj:

Qhov hnyav 22.8g K₂HPO₄.3 H₂O rau 1L ntawm deionized dej kom yaj.

Kev daws 3: 1M HCl tov

Dissolve 8.3ml Concentrated hydrochloric acid nrog dej deionized rau 100 ml.

Txoj Kev 4:1M NaOH solution

Dissolve 4g NaOH nrog 100ml deionized dej.

Kev daws 5: Extraction tov:

Dilute 2 × concentrated extraction tov nrog dej deionized nyob rau hauv lub ntim piv ntawm 1: 1.Qhov kev daws teeb meem no tuaj yeem khaws cia rau 1 lub hlis ntawm 4 ℃, uas yuav raug siv los muab cov qauv coj mus kuaj.

Kev daws 6: wash solution:

Dilute 20 × concentrated ntxuav tov nrog dej deionized nyob rau hauv lub ntim ntawm 1:19, uas yuav siv tau los ntxuav daim hlau.Cov tshuaj diluted no tuaj yeem khaws cia rau 1 lub hlis ntawm 4 ℃.

1. 8.Kev npaj ua qauv

8.1Daim ntawv ceeb toom thiab ceev faj ua ntej ua haujlwm:

a) Thov siv cov lus qhia ib zaug hauv qhov kev sim, thiab hloov cov lus qhia thaum nqus cov reagent sib txawv.

b) Xyuas kom meej tias tag nrho cov cuab yeej sim tau huv.

c) derivative reagent tuaj yeem khaws cia ntawm 2-8 ℃ rau peb lub hlis;

d) Cov tshuaj HCl tuaj yeem khaws cia hauv chav sov li 3 lub hlis;

e) Cov tshuaj NaOH tuaj yeem khaws cia rau 3 lub hlis ntawm chav tsev kub;

f) Khaws cov qauv uas tsis kho rau hauv khov (-20 ℃);

g) Cov qauv kho tuaj yeem khaws cia rau 24h ntawm 2-8 ℃ hauv qhov tsaus ntuj.

8.2 Tsiaj cov ntaub so ntswg thiab lub siab kuaj:

---- Homogenize cov qauv nrog homogenizer;

---- Nyhav 1.0 ± 0.05g ntawm cov ntaub so ntswg homogenized rau hauv 50ml polystyrene centrifuge raj.Ntxiv 4ml deionized dej, 0.5ml 1M HCl tov thiab 100ul derivative reagent (saib tshuaj 1).Co nws rau 2 feeb.

---- Incubate ntawm 37 ℃ thaum hmo ntuj (kwv yees li 16 teev);

---- Ntxiv 5ml 0.1MK₂HPO₄ (daws2), 0.4ml 1M NaOH (daws4) thiab 5ml ethyl acetate.Ua kom nruj rau 30s;

---- Centrifuge ntawm chav tsev kub (20-25 ℃) rau 10min, yam tsawg 3000g;

---- Siv 2.5ml ntawm supernatant organic theem rau hauv 10ml huv iav raj, qhuav nrog 50-60 ℃ nitrogen gas los yog teb evaporator;

---- Dissolve cov khoom seem qhuav nrog 1ml n-hexane (los yog n- heptane), vortex rau 30s, ntxiv 1ml extraction tov (daws5), vortex 1min, sib tov tag nrho.

---- Centrifuge ntawm chav tsev kub (20-25^oC) rau 5 feeb, tsawg kawg yog 3000g;

---- Tshem tawm cov theem supernatant organic;noj 50μl ntawm lub substrate dej theem rau kev soj ntsuam.

8.4 Zoo

---- hnyav 1.0 ± 0.05g ntawm homogenized zib ntab qauv rau hauv 50ml polystyrene centrifuge raj;

---- Ntxiv 4ml deionized dej, 0.5ml 1M HCl (daws3) thiab 100μl derivative reagent (daws1);co tag nrho rau 2 min;

----Incubate ntawm 37 ℃ thaum hmo ntuj (kwv yees li 16 teev);

---- Ntxiv 5ml 0.1MK₂HPO₄ (daws2), 0.4ml 1M NaOH (daws4) thiab 5ml ethyl acetate, co hnyav rau 30s;

---- Centrifuge ntawm chav tsev kub (20-25 ℃) rau 10min, tsawg kawg yog 3000g;

---- Siv 2.5ml ntawm supernatant organic theem rau hauv 10ml huv iav raj, qhuav nrog 50-60 ℃ nitrogen gas los yog teb evaporator;

---- Dissolve lub qhuav seem nrog 1ml n-hexane (los yog n- heptane), vortex rau 30s, ntxiv 1ml extraction tov (daws5), vortex 1min, sib tov tag nrho.

---- Centrifuge ntawm chav tsev kub (20-25^oC) rau 10min, yam tsawg 3000g;

---- Tshem tawm cov theem supernatant organic;noj 50μl ntawm lub substrate dej theem rau kev soj ntsuam.

1. 9.txheej txheem xeem

9.1Daim ntawv ceeb toom ua ntej kev xeem

9.1.1 Nco ntsoov tias tag nrho cov reagents thiab microwells nyob rau hauv chav tsev kub (20-25 ℃).

9.1.2 Rov qab tag nrho cov seem reagents rau 2-8 ℃ tam sim tom qab siv.

9.1.3 Ntxuav lub 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.

9.1.4 Zam lub teeb thiab npog lub microwells thaum lub sij hawm 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, homogenize 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 Cov tshuaj ntxuav cov concentrated thiab cov concentrated extraction tov 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 ibDilution ntawm concentrated antibody tov: dilute cov concentrated enzyme tov nrog enzyme conjugate diluents nyob rau hauv lub ntim piv ntawm 1:10 (1 quav concentrated enzyme tov: 10 folds enzyme conjugate diluents).

9.2.6 ibNtxiv cov qauv tshuaj / qauv thiab enzyme conjugate tov: ntxiv 50µl ntawm cov qauv kev daws teeb meem lossis cov qauv npaj rau cov qhov dej sib thooj, ntxiv 50µl enzyme conjugate tov.Sib tov maj mam los ntawm co lub phaj manually thiab incubate rau 30min ntawm 25 ℃ nrog npog.

9.2.7 ibNtxuav: Tshem tawm lub hau npog maj mam hliv cov dej tawm ntawm qhov dej thiab yaug cov microwells nrog 250µl diluted ntxuav kua (daws6) 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).

9.2.8 ibXim xim: Ntxiv 50µl substrate tov A thiab 50ul substrate tov B rau txhua qhov dej.Sib tov maj mam los ntawm kev co lub phaj manually thiab incubate rau 15 feeb ntawm 25 ℃ nrog npog (saib 12.8).

9.2.11 ibNtsuas: Ntxiv 50µl cov tshuaj nres rau txhua qhov dej.Sib tov maj mam los ntawm kev co lub phaj manually thiab ntsuas qhov absorbance ntawm 450nm (Nws 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

10.1Feem 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 —— nqus tus qauv (los yog qauv)

B0 —— absorbance zero standard

10.2Standard Curve

---- Txhawm rau kos tus qauv nkhaus: Siv tus nqi nqus ntawm cov qauv raws li y-axis, ib nrab logarithmic ntawm qhov concentration ntawm AOZ tus qauv tov (ppb) raws li x-axis.

---- Lub AOZ 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 ntawm txhua tus qauv ua raws, thiab qhov tseeb concentration ntawm cov qauv tau txais.

Thov ceeb toom:

Cov software tshwj xeeb tau tsim rau txhua qhov kev txo cov ntaub ntawv, uas tuaj yeem muab tau raws li kev thov.

Dilution factor………………………………………………………………… 2

10.rhiab heev, raug thiab precision

rhiab heev: 0.025 wb

Kev kuaj pom qhov txwv:

Tissue (muscle, siab)…………………………0.1ppb

Honey--------------------------------------ua tsaug -0.1 Ib

Qhov tseeb:

Tsiaj cov ntaub so ntswg (cov leeg nqaij thiab lub siab) ………………… 100 ± 20%

Hniav ………………………………..………………………… 100 ± 20%

Precision:CV ntawm cov khoom siv ELISA tsawg dua 10%.

11.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 Cia txias: 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 yuav deteriorated yog tias tus nqi absorbance (450/630nm) ntawm tus qauv xoom tsawg dua 0.5 (A450nm <0.5).

12.8 Cov tshuaj tiv thaiv xim xav tau 15 feeb tom qab ntxiv cov tshuaj substrate;Koj tuaj yeem ncua lub sijhawm incubation mus rau 20min lossis ntau dua yog tias cov xim yog lub teeb dhau los txiav txim siab., tsis txhob dhau 25min.On qhov tsis sib xws, luv lub sijhawm incubation kom zoo.

12.9 Qhov zoo tshaj plaws tshuaj tiv thaiv kub yog 25 ℃.Kev kub siab los yog qis dua yuav ua rau muaj kev hloov pauv ntawm rhiab heev thiab absorbance qhov tseem ceeb.

12.Cia txias thiab lub sijhawm cia

Cia txias: 2-8 ℃.

Lub sijhawm cia: 12 lub hlis.