Idije Enzyme Immunoassay Kit fun Itupalẹ pipo ti Furazolidone metabolite (AOZ)
- 1.abẹlẹ
Nitrofurans jẹ awọn aporo apanirun ti o gbooro sintetiki, eyiti o jẹ iṣẹ nigbagbogbo ni iṣelọpọ ẹranko fun awọn ohun-ini antibacterial ti o dara julọ ati awọn ohun-ini elegbogi.Wọn tun ti lo bi awọn olupolowo idagbasoke ni ẹlẹdẹ, adie ati iṣelọpọ omi.Ninu awọn iwadii igba pipẹ pẹlu awọn ẹranko laabu fihan pe awọn oogun obi ati awọn metabolites wọn ṣe afihan carcinogenic ati awọn abuda mutagenic.Eyi ti yori si idinamọ ti nitrofurans fun itọju awọn ẹranko ti a lo fun iṣelọpọ ounjẹ.Awọn oogun nitrofuran furaltadone, nitrofurantoin ati nitrofurazone ni a fi ofin de lilo ninu iṣelọpọ ẹran ni EU ni ọdun 1993, ati lilo furazolidone ti ni idinamọ ni ọdun 1995.
Onínọmbà ti awọn iyokuro awọn oogun nitrofuran nilo lati da lori wiwa ti awọn metabolites ti a so mọ ti awọn oogun obi nitrofuran.Niwọn igba ti awọn oogun obi ti ni iṣelọpọ ni iyara pupọ, ati awọn metabolites nitrofuran ti ara ti o ni asopọ yoo da duro fun igba pipẹ, awọn metabolites wọnyi ni a lo bi ibi-afẹde ni wiwa ilokulo ti nitrofurans, eyiti o pẹlu Furazolidone metabolite (AOZ), metabolite Furaltadone (AMOZ). ), Nitrofurantoin metabolite (AHD) ati Nitrofurazone metabolite (SEM).
Awọn iṣẹku AOZ jẹ ipinnu pupọ julọ nipasẹ LC-MS tabi LC-MS/MS.Enzyme immunoassays, ni akawe pẹlu awọn ọna chromatographic, ṣafihan awọn anfani pupọ nipa ifamọ, opin wiwa, ohun elo imọ-ẹrọ ati ibeere akoko.( Iye owo akoko: iṣẹju 45)
- 2.Ilana Idanwo
Ohun elo ELISA yii jẹ apẹrẹ lati ṣe awari AOZ ti o da lori ipilẹ ti immunoassay enzyme aiṣe-idije.Awọn kanga microtiter jẹ ti a bo pẹlu gbigba antijeni ti o ni asopọ BSA.AOZ ni apẹẹrẹ ti njijadu pẹlu antijeni ti a bo lori awo microtiter fun aporo ti a ṣafikun.Lẹhin afikun ti enzymu conjugate, a lo sobusitireti chromogenic ati pe ifihan naa jẹ iwọn nipasẹ spectrophotometer kan.Imudani jẹ iwọn idakeji si ifọkansi AOZ ninu apẹẹrẹ.
- 3.Awọn ohun elo
Ohun elo yii le ṣee lo ni pipo ati igbekale agbara ti iyoku AOZ nianima tissues(isan, ẹdọ ati bẹbẹ lọ), oyin.
- 4.Cross-aati
Furazolidone metabolite (AOZ) ………………………………….. 100%
Furaltadone metabolite (AMOZ) …………………………………………….
Nitrofurantoin metabolite (AHD) …………………………………………………….
Nitrofurazone metabolite (SEM) …………………………………………………………….
Furazolidone …………………………………………………………………………………………………………………
Furaltadone …………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………….
Nitrofurantoin……………………………………………………………………….. <1%
Nitrofurazone …………………………………………………………………………………………………………………………………………
- 5.Awọn ohun elo ti a beere
5.1Awọn ohun elo
spectrophotometer awo Microtiter (450nm/630nm)
----Iyipo evaporator tabi awọn ohun elo gbigbe nitrogen
----Homogenizer /stomacher
----Shaker
----Apopọ Vortex
----Centrifuge
---- Iwọntunwọnsi itupalẹ (inductance: 0.01g)
---- pipette ti o gboye: 10ml
---- Roba pipette boolubu
----Ala igo: 100ml
---- Gilasi gilasi: 10ml
----Polystyrene centrifuge tube: 2ml, 50ml
----Micropipettes: 20ul-200ul, 100ul-1000ul,
250ul-multipipette
5.2Reagents
----Ethyl acetate (AR)
----n-hexane (tabi n-heptane) (AR)
----Dipotassium hydrogen fosifeti trihydrate
(K2HPO4.3H2O) (AR)
----Hydrochloric acid ti o ni idojukọ (HCl, AR)
--Methanol
---- Sodium hydroxide (NaOH, AR)
----Omi ti a fi omi ṣan
- 6.Ohun elo Kit
l Microtiter awo ti a bo pẹlu antijeni, 96 kanga
l Awọn ojutu boṣewa (igo 6, 1 milimita / igo)
0ppb,0.025ppb,0.075ppb,0.225ppb,0.675ppb,2.025ppb
l Spiking boṣewa Iṣakoso: (1ml/igo).......100ppb
l Enzymu ogidi conjugate 1ml ...... fila pupa
l Enzyme conjugate diluents 10ml……….. fila alawọ ewe
l Sobusitireti A 7ml ……………………………………………………….. fila funfun
l Sobusitireti B 7ml……………………………………………………….. fila pupa
l Duro ojutu 7ml……………………………………… fila ofeefee
l 20× ogidi w ojutu 40ml
…………………………………………………………………………………………………. fila sihin
l 2× ojutu isediwon ogidi 60ml….bulu fila
l 2-Nitrobenzaldehyde 15.1mg……………… fila funfun
- 7.Igbaradi Reagents
Ojutu 1: reagent itọsẹ:
Fi 10ml ti kẹmika kẹmika si igo ti o ni 2-Nitrobenzaldehyde ati tu.(ni ifọkansi ti 10mM).
Ojutu 2: 0.1MK2HPO4ojutu:
Ṣe iwuwo 22.8g K2HPO4.3H2O si 1L ti omi deionized lati tu.
Ojutu 3: 1M HCl ojutu
Tu 8.3ml Ogidi hydrochloric acid pẹlu deionized omi to 100ml.
Ojutu 4:1M NaOH ojutu
Tu 4g NaOH pẹlu omi diionized 100ml.
Ojutu 5: ojutu isediwon:
Dilute 2 × ojutu isediwon ogidi pẹlu omi deionized ni ipin iwọn didun ti 1: 1.Ojutu yii le ṣe itọju fun oṣu 1 ni 4℃, eyiti yoo ṣee lo lati fa awọn ayẹwo jade.
Ojutu 6: ojutu fifọ:
Din 20 × ogidi fifọ ojutu pẹlu omi deionized ni iwọn didun 1:19, eyi ti ao lo lati wẹ awọn awopọ.Ojutu ti fomi le wa ni ipamọ fun oṣu kan ni 4℃.
- 8.Apeere Igbaradi
8.1Akiyesi ati awọn iṣọra ṣaaju ṣiṣe:
a) Jọwọ lo ọkan-pipa awọn imọran ninu awọn ṣàdánwò, ki o si yi awọn italologo nigba ti absorbing o yatọ si reagent.
b) Rii daju pe gbogbo awọn ohun elo idanwo jẹ mimọ.
c) reagent itọsẹ le wa ni fipamọ ni 2-8 ℃ fun oṣu mẹta;
d) Ojutu HCl le wa ni ipamọ ni iwọn otutu yara fun oṣu mẹta;
e) Ojutu NaOH le wa ni ipamọ fun awọn oṣu 3 ni iwọn otutu yara;
f) Jeki awọn ayẹwo ti ko ni itọju ni didi (-20 ℃);
g) Awọn ayẹwo ti a ṣe itọju le ṣe itọju fun wakati 24 ni 2-8℃ ninu okunkun.
8.2 Ẹranko ẹran ati awọn ayẹwo ẹdọ:
----Homogenize awọn ayẹwo pẹlu homogenizer;
----Iwọn 1.0 ± 0.05g ti ayẹwo ara homogenized sinu tube centrifuge polystyrene 50ml.Fi 4ml deionized omi, 0.5ml 1M HCl ojutu ati 100ul itọsẹ reagent (wo ojutu 1).Gbọn fun iṣẹju 2.
---- Incubate ni 37 ℃ moju (nipa 16h);
---- Fi 5ml 0.1MK kun2HPO4 (ojutu2), 0.4ml 1M NaOH (ojutu4) ati 5ml ethyl acetate.Gbọn lile fun 30s;
---- Centrifuge ni iwọn otutu yara (20-25 ℃) fun iṣẹju 10, o kere ju 3000g;
---- Mu 2.5ml ti ipele Organic supernatant sinu tube gilasi mimọ 10ml, gbẹ pẹlu gaasi nitrogen 50-60℃ tabi evaporator rotari;
--- Tu ajẹkù ti o gbẹ pẹlu 1ml n-hexane (tabi n-heptane), vortex fun 30s, ṣafikun ojutu isediwon 1ml (ojutu5), vortex 1min, dapọ patapata.
---- Centrifuge ni otutu yara (20-25oC) fun iṣẹju 5, o kere ju 3000g;
---- Yọ awọn supernatant Organic alakoso;mu 50μl ti ipele omi sobusitireti fun ayẹwo.
8.4 Oyin
--- ṣe iwọn 1.0 ± 0.05g ti apẹẹrẹ oyin homogenized sinu tube centrifuge polystyrene 50ml;
---- Ṣafikun 4ml omi ti a ti sọ diionized, 0.5ml 1M HCl (ojutu3) ati 100μl itọsẹ reagent (ojutu1);gbọn patapata fun iṣẹju 2;
----Incubate ni 37℃ moju (nipa 16h);
----Fi 5ml 0.1MK kun2HPO4 (ojutu2), 0.4ml 1M NaOH (ojutu4) ati 5ml ethyl acetate, gbigbọn fifẹ fun 30s;
----Centrifuge ni iwọn otutu yara (20-25 ℃) fun iṣẹju 10, o kere ju 3000g;
---- Mu 2.5ml ti ipele Organic supernatant sinu tube gilasi mimọ 10ml, gbẹ pẹlu gaasi nitrogen 50-60℃ tabi evaporator rotari;
---- Tu ajẹkù ti o gbẹ pẹlu 1ml n-hexane (tabi n-heptane), vortex fun 30s, ṣafikun ojutu isediwon 1ml (ojutu5), vortex 1min, dapọ patapata.
----Centrifuge ni otutu yara (20-25oC) fun iṣẹju 10, o kere ju 3000g;
----Yọ awọn supernatant Organic alakoso;mu 50μl ti ipele omi sobusitireti fun ayẹwo.
- 9.Ilana ayẹwo
9.1Ṣe akiyesi ṣaaju ayẹwo
9.1.1 Rii daju pe gbogbo reagents ati microwells wa ni gbogbo ni yara otutu (20-25 ℃).
9.1.2 Pada gbogbo awọn reagents isinmi pada si 2-8 ℃ lẹsẹkẹsẹ lẹhin lilo.
9.1.3 Fifọ awọn microwells ti o tọ jẹ igbesẹ pataki ninu ilana ti iṣiro;o jẹ ifosiwewe pataki si isọdọtun ti itupalẹ ELISA.
9.1.4 Yago fun ina ati ki o bo awọn microwells nigba abeabo.
9.2 Assay Igbesẹ
9.2.1 Ya gbogbo reagents jade ni yara otutu (20-25 ℃) fun diẹ ẹ sii ju 30min, homogenize ṣaaju lilo.
9.2.2 Gba awọn microwells ti o nilo jade ki o da iyoku pada sinu apo titiipa zip ni 2-8℃ lẹsẹkẹsẹ.
9.2.3 Ojutu fifọ ifọkansi ati ojutu isediwon ifọkansi yẹ ki o tun wa ni iwọn otutu ṣaaju lilo.
9.2.4Nọmba:Nọmba gbogbo awọn ipo microwell ati gbogbo awọn iṣedede ati awọn ayẹwo yẹ ki o ṣiṣẹ ni ẹda-ẹda.Ṣe igbasilẹ awọn iṣedede ati awọn ipo apẹẹrẹ.
9.2.5Dilution ti ogidi antibody ojutu: dilute the ogidi henensiamu ojutu pẹlu henensiamu conjugate diluents ni awọn iwọn didun ipin ti 1:10(1 agbo ogidi enzymu ojutu: 10 folds enzymu conjugate diluents).
9.2.6Ṣafikun ojutu boṣewa / apẹẹrẹ ati ojutu conjugate henensiamu: ṣafikun 50µl ti ojutu boṣewa tabi apẹẹrẹ ti a pese silẹ si awọn kanga ti o baamu, ṣafikun ojutu conjugate enzyme 50µl.Illa rọra nipa gbigbọn awo pẹlu ọwọ ati incubate fun 30min ni 25℃ pẹlu ideri.
9.2.7Fọ: Yọ ideri naa rọra ki o si tú omi jade kuro ninu awọn kanga ki o fi omi ṣan awọn microwells pẹlu 250µl ti fomi ojutu (250µl).ojutu6) ni aarin ti 10s fun 4-5 igba.Fa omi to ku pẹlu iwe ifamọ (okuta afẹfẹ iyokù le yọkuro pẹlu imọran ti ko lo).
9.2.8AwọṢafikun ojutu sobusitireti 50µl A ati ojutu sobusitireti 50ul B si kanga kọọkan.Darapọ mọra nipa gbigbọn awo pẹlu ọwọ ati incubate fun iṣẹju 15 ni 25℃ pẹlu ideri (wo 12.8).
9.2.11IwọnFi 50µl ojutu iduro si kanga kọọkan.Darapọ ni rọra nipa gbigbọn awo pẹlu ọwọ ati wiwọn ifasilẹ ni 450nm (O daba wiwọn pẹlu gigun-meji ti 450/630nm. Ka abajade laarin iṣẹju 5 lẹhin afikun ti ojutu iduro.)
10 Awọn abajade
10.1Gbigba ogorun
Awọn iye iwọn ti awọn iye ifunmọ ti a gba fun awọn iṣedede ati awọn apẹẹrẹ ti pin nipasẹ iye gbigba ti boṣewa akọkọ (boṣewa odo) ati isodipupo nipasẹ 100%.Iwọnwọn odo jẹ bayi jẹ dogba si 100% ati awọn iye gbigba ni a sọ ni awọn ipin ogorun.
=
B ——boṣewa gbigba (tabi apẹẹrẹ)
B0 ——absorbance odo bošewa
10.2Standard ti tẹ
----Lati fa a boṣewa ti tẹ: Mu absorbance iye ti awọn ajohunše bi y-axis, ologbele logarithmic ti awọn fojusi ti AOZ boṣewa ojutu (ppb) bi x-apa.
---- Idojukọ AOZ ti ayẹwo kọọkan (ppb), eyiti o le ka lati inu ọna kika, ti wa ni isodipupo nipasẹ iṣiro dilution ti o baamu ti ayẹwo kọọkan ti o tẹle, ati pe a gba ifọkansi gangan ti ayẹwo.
Jọwọ ṣe akiyesi:
Sọfitiwia pataki ti ni idagbasoke fun gbogbo idinku data, eyiti o le pese lori ibeere.
Dilution ifosiwewe…………………………………………………………………………………………………
10.Ifamọ, išedede ati konge
Ifamọ: 0.025ppb
Iwọn wiwa:
Tissue (isan, ẹdọ) ………………………………………….0.1ppb
Oyin--------------------------------- -0.1ppb
Yiye:
Ẹran ẹran ara (isan ati ẹdọ) ………………………………………………………… 100± 20%
Oyin………………………………………………………………………………. 100±20%
Itọkasi:CV ti ohun elo ELISA ko kere ju 10%.
11.Akiyesi
12.1 Awọn iye iwọn ti awọn iye ifunmọ ti a gba fun awọn iṣedede ati awọn ayẹwo yoo dinku ti awọn reagents ati awọn ayẹwo ko ba ti ni ilana si iwọn otutu yara (20-25 ℃).
12.2 Ma ṣe jẹ ki awọn microwells gbẹ laarin awọn igbesẹ lati yago fun atunṣe aṣeyọri ati ṣiṣẹ ni igbesẹ ti n tẹle lẹsẹkẹsẹ lẹhin ti o tẹ ohun mimu microwells.
12.3 Gbọn kọọkan reagent rọra ṣaaju lilo.
12.4 Jeki awọ ara rẹ kuro ni ojutu iduro fun o jẹ 0.5MH2SO4ojutu.
12.5 Maṣe lo awọn ohun elo ti ọjọ.Maṣe paarọ awọn reagents ti awọn ipele oriṣiriṣi, bibẹẹkọ yoo ju ifamọ naa silẹ.
12.6 Ipo ipamọ: Jeki awọn ohun elo ELISA ni 2-8℃, ma ṣe di.Di awọn apẹrẹ microwell isinmi, Yago fun imọlẹ orun taara lakoko gbogbo awọn ifisi.Ibora awọn apẹrẹ microtiter ni a ṣe iṣeduro.
12.7 Ojutu sobusitireti yẹ ki o kọ silẹ ti o ba yi awọn awọ pada.Awọn reagents le bajẹ ti iye gbigba (450/630nm) ti boṣewa odo jẹ kere ju 0.5 (A450nm <0.5).
12.8 Idahun awọ nilo 15min lẹhin afikun ti ojutu sobusitireti;O le fa akoko idabobo naa pọ si iṣẹju 20 tabi diẹ sii ti awọ ba jẹ ina pupọ lati pinnu., Maṣe kọja 25min. Ni ilodi si, kuru akoko isunmọ daradara.
12.9 Awọn iwọn otutu lenu ti o dara julọ jẹ 25 ℃.Iwọn giga tabi isalẹ yoo yorisi awọn iyipada ti ifamọ ati awọn iye gbigba.
12.Ipo ipamọ ati akoko ipamọ
Ipo ipamọ: 2-8℃.
Akoko ipamọ: 12 osu.
