khoom

Test Principple

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.Flumequineresidue 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 tsis zoo cuam tshuam nrog cov tetracycline nyob hauv nws, tom qab muab piv nrog Standard Curve, muab faib los ntawm ntau yam dilution, Flumequine residue kom muaj nuj nqis hauv cov qauv tuaj yeem xam.

Daim ntawv thov

Cov khoom siv no tuaj yeem siv rau hauv kev ntsuas ntau thiab zoo ntawm flumequine residue hauv zib ntab.

Kev sib tshuam

Flumequine ………………………………………………………… 100%

Cov ntaub ntawv xav tau

Cov khoom siv

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

┅┅ Homogenizer los yog lub plab

Nws yog ib qho tsim nyog yuav tau siv lub tshuab ntxhua khaub ncaws Shaker

┅┅Vortex tov khoom

Lub tshuab nqus tsev twj tso kua mis centrifuge

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

┅┅ Graduated pipette: 15ml

┅┅ Rubber pipette noob

┅┅ Polystyrene Centrifuge tube: 15ml, 50ml

┅┅ iav raj: 10ml

┅┅ Micropipettes: 20ml-200ml, 100ml-10000ml,

250 ml - ntau yam

Reagents

┅┅n-hexane (AR)

┅┅ Methylene chloride (AR)

┅┅Acetonitrile (AR)

┅┅ Deionized dej

----- Concentrated hydrochloric acid (AR)

Cov khoom siv

● Microtiter phaj nrog 96 qhov dej coated nrog antigen

● Cov kev daws teeb meem (6 fwj × 1ml / lub raj mis)

0 ppb, 0.3 ppb, 1.2 ppb, 4.8 ppb, 19.2 ppb, 76.8 ppb

● High concentration standard control: (1ml / lub raj mis)

………………………………………………………………….100 ppb

● Enzyme conjugate 12ml………………………... liab cap

● Antibody tov 7ml …………..……..…..

● Tshuaj A 7ml……..…………………..………..White cap

● Tshuaj B 7ml …………….…………..…………… liab cap

● Stop solution 7ml …………………………..……… daj hau

● 20XConcentrated ntxuav kua 40ml

…………………………………………………….. Transparent cap

●2X Extraction tov 50ml………………..… xiav cap

Kev npaj Reagents

7.1 Honey qauv

Kev daws 1: 0.2 M Hydrochloric acid tov

Qhov hnyav 41.5ml Concentrated hydrochloric acid, dilute nrog dej deionized rau 500 ml.

Kev daws 2: Ntxuav cov tshuaj

Dilute cov tshuaj ntxuav kom huv nrog dej deionized hauv qhov sib piv ntawm 1:19, uas yuav siv rau ntxuav cov phiaj.cov tshuaj diluted tuaj yeem khaws cia ntawm 4 ℃ rau 1 lub hlis.

Kev daws3: kev daws teeb meem

Dilute 2 × concentrated extraction tov nrog dej deionized nyob rau hauv lub ntim ntawm 1: 1 (los yog nyob ntawm seb yuav tsum tau), uas yuav siv tau rau cov qauv extraction.Cov tshuaj diluted no tuaj yeem khaws cia rau 1 lub hlis ntawm 4 ℃.

Kev npaj ua qauv

8.1 Daim ntawv ceeb toom thiab ceev faj rau cov neeg siv 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 tag nrho cov cuab yeej sim ua kom huv si, txwv tsis pub nws yuav cuam tshuam qhov kev xeem.

8.2Honey qauv

----- Nyhav 2g ± 0.05g zib ntab qauv rau hauv 50ml polystyrene centrifuge raj,

----- Ntxiv 2ml 0.2 M Hydrochloric acid tov (Kev daws teeb meem 1), vortex kom nws tag, ces ntxiv 8ml methylene chloride, co nrog shaker rau 5min kom yaj tag;

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

----- Tshem tawm cov theem supernatant, noj 2 ml ntawm substrate organic tov rau 10 ml iav raj.dry lub substate nyob rau hauv dej da dej ntawm Nitrogen txaus (50-60 ℃)

----- Ntxiv 1 ml n-hexane, vortex rau 30s, ces ntxiv 1ml extraction tov (xov 3), vortex dua rau 1min.centrifuge rau 5min, tsawg kawg yog 3000g ntawm chav tsev kub (20-25 ℃);

----- Tshem tawm cov theem supernatant, noj 50ml rau kev soj ntsuam;

9. Kev ntsuam xyuas

9.1 Daim ntawv ceeb toom ua ntej kev ntsuam xyuas

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 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 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 ibNtxiv cov qauv kev daws teeb meem / qauv:Ntxiv 50 µl ntawm cov qauv kev daws teeb meem lossis cov qauv npaj rau cov qhov dej sib thooj.Ntxiv 50µl tshuaj tiv thaiv kab mob.Sib tov maj mam los ntawm co lub phaj manually thiab incubate rau 30min ntawm 25 ℃ nrog npog.

9.2.6 ibNtxuav:Tshem tawm lub hau npog maj mam thiab ntxuav cov kua dej tawm ntawm qhov dej thiab yaug cov microwells nrog 250µl diluted wash solution (xov 2) 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 ib.Enzyme conjugate:Ntxiv enzyme conjugate tov 100ml rau txhua lub qhov dej, Sib tov maj mam los ntawm co lub phaj manually thiab incubate rau 30min ntawm 25 ℃ nrog npog.Rov ua kauj ruam ntxuav dua.

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

9.2.9 ibNtsuas:Ntxiv 50µl cov tshuaj nres rau txhua qhov dej.Sib tov maj mam los ntawm co lub phaj manually thiab ntsuas qhov nqus ntawm 450nm tiv thaiv huab cua dawb paug (Nws tau pom zoo ntsuas nrog dual-wavelength ntawm 450/630nm. Nyeem qhov tshwm sim hauv 5min tom qab ntxiv cov tshuaj nres.) (Peb kuj tuaj yeem ntsuas los ntawm kev pom. tsis muaj kev daws teeb meem luv luv ntawm ELIASA ntsuas)

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.

Absorbance (%) = B/B0 × 100%

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, semi logarithmic ntawm qhov concentration ntawm flumequine cov qauv tshuaj (ppb) raws li x-axis.

--- Covflumequineconcentration ntawm txhua tus qauv (ppb), uas tuaj yeem nyeem tau los ntawm kev ntsuas qhov nkhaus, yog muab faib los ntawm qhov sib txuam dilution ntau ntawm txhua tus qauv ua raws, thiab qhov tseeb concentration ntawm cov qauv tau txais.

Rau cov ntaub ntawv txo cov khoom siv ELISA, software tshwj xeeb tau tsim, uas tuaj yeem muab tau raws li kev thov.

11. rhiab heev, raug thiab precision

Test Sensitivity:0,3p wb

Honey Sample dilution factor: 2

Kev kuaj pom txwv

Honey qauv--------------------------------- -1 ppb

Qhov tseeb

Honey qauv ------------------------------------------------ 90 ± 20 %

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 rov qab thiab ua haujlwm cov kauj ruam tom ntej tam sim tom qab coj mus rhaub lub microwells.

12.3.Homogenize txhua reagent ua ntej siv.

12.4.Khaws koj cov tawv nqaij kom deb ntawm qhov kev daws teeb meem rau nws yog 2M H₂SO₄daws.

12.5 Tsis txhob siv cov khoom siv dhau los.Tsis txhob pauv cov reagents ntawm cov khoom sib txawv, vim nws yuav poob qhov rhiab heev.

12.6 Storage condition:

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 Indications rau reagents mus phem:

Cov tshuaj substrate 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 tshuaj A thiab tshuaj B. Thiab koj tuaj yeem ncua lub sijhawm incubation ntawm 20min mus rau ntau dua yog tias cov xim teeb dhau los txiav txim siab.Tsis txhob tshaj 25min, Ntawm qhov tsis sib xws, txo lub sij hawm 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.

13. Cia

Cia txias: 2-8 ℃.

Lub sijhawm cia: 12 lub hlis.