Gasar Enzyme Immunoassay Kit don ƙididdige ƙididdigar Furazolidone metabolite (AOZ)
- 1.Fage
Nitrofurans maganin rigakafi ne na roba mai faɗi, waɗanda akai-akai ana aiki da su a cikin samar da dabbobi don kyawawan abubuwan kashe ƙwayoyin cuta da abubuwan pharmacokinetic.An kuma yi amfani da su azaman masu haɓaka haɓakar alade, kiwon kaji da samar da ruwa.A cikin dogon lokaci bincike tare da dabbobin lab ya nuna cewa magungunan iyaye da metabolites ɗin su sun nuna halayen carcinogenic da mutagenic.Wannan ya haifar da haramcin nitrofuran don kula da dabbobin da ake amfani da su don samar da abinci.An haramta amfani da magungunan nitrofuran furaltadone, nitrofurantoin da nitrofurazone daga amfani da su wajen samar da dabbobi a cikin EU a cikin 1993, kuma an hana amfani da furazolidone a cikin 1995.
Binciken ragowar magungunan nitrofuran yana buƙatar dogara ne akan gano ƙwayoyin da aka ɗaure nama na magungunan iyaye na nitrofuran.Tun da magungunan iyaye suna da sauri sosai, kuma nama da ke daure nitrofuran metabolites zai riƙe na dogon lokaci, ana amfani da waɗannan metabolites a matsayin manufa a cikin gano cin zarafi na nitrofuran, wanda ya haɗa da Furazolidone metabolite (AOZ), Furaltadone metabolite (AMOZ). Nitrofurantoin metabolite (AHD) da Nitrofurazone metabolite (SEM).
AOZ-ragowar an ƙaddara mafi yawanci ta LC-MS ko LC-MS/MS.Enzyme immunoassays, idan aka kwatanta da chromatographic hanyoyin, nuna babba abũbuwan amfãni game da hankali, iyakan ganewa, fasaha kayan aiki da kuma lokaci da ake bukata.(Farashin lokacin: 45min)
- 2.Ƙa'idar Gwaji
An tsara wannan kit ɗin ELISA don gano AOZ bisa ka'idar immunoassay enzyme mai gasa kai tsaye.An lulluɓe rijiyoyin microtiter tare da kama antigen mai alaƙa da BSA.AOZ a cikin samfurin yana gasa tare da antigen da aka rufa akan farantin microtiter don ƙarar rigakafin.Bayan da aka ƙara enzyme conjugate, ana amfani da substrate na chromogenic kuma ana auna siginar ta hanyar spectrophotometer.Abun sha ya bambanta da daidaituwar AOZ a cikin samfurin.
- 3.Aikace-aikace
Ana iya amfani da wannan kit ɗin a ƙididdigewa da ƙididdige ƙididdiga na ragowar AOZ a cikianima tissues( tsoka, hanta da sauransu), zuma.
- 4.Maganganun ra'ayi
Furazolidone metabolite (AOZ)……………………………………………….
Furaltadone metabolite (AMOZ) …………………………………………………………….
Nitrofurantoin metabolite (AHD)……………………………… <0.1%
Nitrofurazone metabolite (SEM)………………………………………………………………………….
Furazolidone ……………………………………………………………………………………………………………………………………
Furaltadone………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………….
Nitrofurantoin………………………………………………………………………………………………………………………………………………………………………………………….
Nitrofurazone………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………….
- 5.Abubuwan da ake buƙata
5.1Kayan aiki
----Microtiter farantin spectrophotometer (450nm/630nm)
---- Rotary evaporator ko kayan bushewar nitrogen
----Homogenizer / ciki
----Shakar
---- Mai haɗa Vortex
----Centrifuge
---- Ma'aunin nazari (inductance: 0.01g)
----Piette da aka sauke: 10ml
---- Rubber pipette kwan fitila
----Filin girma: 100ml
---- Gilashin gilashi: 10ml
----Polystyrene centrifuge tube: 2ml, 50ml
----Micropipettes: 20ul-200ul, 100ul-1000ul,
250ul-multipipette
5.2Reagents
----Ethyl acetate (AR)
----n-hexane (ko n-heptane) (AR)
----Dipotassium hydrogen phosphate trihydrate
(K2HPO4.3H2O) (AR)
----Hydrochloric acid mai tattarawa (HCl, AR)
--Methanol
----Sodium hydroxide (NaOH, AR)
---- Ruwan da aka lalata
- 6.Abubuwan Kit
l Farantin Microtiter mai rufi tare da antigen, rijiyoyin 96
l Standard mafita (6 kwalabe, 1ml / kwalban)
0ppb,0.025ppb,0.075ppb,0.225ppb,0.675ppb,2.025ppb
l Sarrafa daidaitaccen iko: (1ml/kwalba).......100ppb
l Ƙarƙashin ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta
l Enzyme conjugate diluents 10ml…………. koren hula
l Substrate A 7ml……………………………………………………………….. farar hula
l Substrate B 7ml………………………………………………………. ja hula
l Tsaida bayani 7ml………………………………………
l 20 × maida hankali wanke bayani 40ml
……………………………………………………………
l 2 × mayar da hankali hakar bayani 60ml…. blue hula
l 2-Nitrobenzaldehyde 15.1mg……………….farin hula
- 7.Shirye-shiryen Reagents
Magani 1Reagent na asali:
Ƙara 10ml na methanol a cikin kwalban yana da 2-Nitrobenzaldehyde kuma narke.(a cikin maida hankali na 10mM).
Magani 2Saukewa: 0.1MK2HPO4mafita:
Nauyin 22.8g K2HPO4.3H2O zuwa 1L na ruwa mai narkewa don narkewa.
Magani 3: 1M HCl bayani
Narkar da 8.3ml Concentrated hydrochloric acid tare da deionized ruwa zuwa 100ml.
Magani 4:1M NaOH bayani
Narkar da 4g NaOH tare da 100ml da aka lalatar da ruwa.
Magani 5Maganin hakar:
Tsarma 2 × mai da hankali hakar bayani tare da deionized ruwa a cikin girma rabo na 1:1.Wannan bayani za a iya kiyaye ga 1month a 4 ℃, wanda za a yi amfani da fitar da samfurori.
Magani 6: maganin wanki:
Tsarma da 20 × mai da hankali bayani mai wankewa tare da ruwa mai tsabta a cikin adadin ƙarar 1:19, wanda za a yi amfani da shi don wanke faranti.Wannan diluted bayani za a iya kiyaye ga 1 watan a 4 ℃.
- 8.Misali Shirye-shiryen
8.1Sanarwa da kariya kafin aiki:
a) Da fatan za a yi amfani da nasihu guda ɗaya a cikin gwaji, kuma canza tukwici yayin ɗaukar reagent daban-daban.
b) Tabbatar cewa duk kayan aikin gwaji suna da tsabta.
c) Za a iya adana reagent mai ƙira a 2-8 ℃ na watanni uku;
d) Ana iya adana maganin HCl a dakin da zafin jiki na watanni 3;
e) Ana iya adana maganin NaOH na tsawon watanni 3 a dakin da zafin jiki;
f) Rike samfurori marasa magani a cikin daskarewa (-20 ℃);
g) Ana iya adana samfuran da aka bi da su don 24h a 2-8 ℃ a cikin duhu.
8.2 Naman dabba da samfuran hanta:
---- Haɗa samfuran tare da homogenizer;
---- Weight 1.0 ± 0.05g na samfurin nama mai kama da shi cikin 50ml polystyrene centrifuge tube.Add 4ml deionized ruwa, 0.5ml 1M HCl bayani da 100ul wanda aka samu reagent (duba bayani 1).Ki girgiza shi na tsawon mintuna 2.
---- Incubate a 37 ℃ na dare (kimanin 16h);
---- Ƙara 5ml 0.1MK2HPO4 (mafita2), 0.4ml 1M NaOH (mafita4) da kuma 5ml ethyl acetate.Girgiza mai zafi don 30s;
---- Centrifuge a dakin da zafin jiki (20-25 ℃) na 10min, akalla 3000g;
---- Ɗauki 2.5ml na lokaci mai girma a cikin bututun gilashi mai tsabta 10ml, bushe tare da 50-60 ℃ nitrogen gas ko rotary evaporator;
---- Narkar da busassun busassun tare da 1ml n-hexane (ko n-heptane), vortex don 30s, ƙara maganin cirewar 1ml (mafita5), vortex 1min, gauraya gaba daya.
---- Centrifuge a dakin da zafin jiki (20-25oC) na 5min, akalla 3000g;
---- Cire mafi girman lokaci;Ɗauki 50 μl na lokaci na ruwa na substrate don tantancewa.
8.4 Ruwa
----nauyin 1.0 ± 0.05g na samfurin zuma na homogenized a cikin 50ml polystyrene centrifuge tube;
---- Add 4ml deionized ruwa, 0.5ml 1M HCl (mafita3) da 100 μl wanda aka samu reagent (mafita1);girgiza gaba daya na 2min;
----Incubate a 37 ℃ na dare (kimanin 16h);
---- Ƙara 5ml 0.1MK2HPO4 (mafita2), 0.4ml 1M NaOH (mafita4) da 5ml ethyl acetate, girgiza sosai don 30s;
----Centrifuge a dakin da zafin jiki (20-25 ℃) na 10min, akalla 3000g;
---- Ɗauki 2.5ml na lokaci mai girma a cikin bututun gilashi mai tsabta 10ml, bushe tare da 50-60 ℃ nitrogen gas ko rotary evaporator;
----Narke busassun busassun tare da 1ml n-hexane (ko n-heptane), vortex don 30s, ƙara maganin cirewar 1ml (mafita5), vortex 1min, gauraya gaba daya.
----Centrifuge a dakin da zafin jiki (20-25oC) na minti 10, akalla 3000g;
---- Cire mafi girman lokaci;Ɗauki 50 μl na lokaci na ruwa na substrate don tantancewa.
- 9.Tsarin tantancewa
9.1Sanarwa kafin tantancewa
9.1.1 Tabbatar cewa duk reagents da microwells duk suna cikin zafin jiki (20-25 ℃).
9.1.2 Koma duk sauran reagents zuwa 2-8 ℃ nan da nan bayan amfani.
9.1.3 Wanke microwells daidai wani muhimmin mataki ne a cikin tsarin tantancewa;Yana da mahimmancin mahimmanci ga sake fasalin binciken ELISA.
9.1.4 Ka guji hasken kuma rufe microwells yayin shiryawa.
9.2 Matakan tantancewa
9.2.1 Ɗauki duk reagents a cikin zafin jiki (20-25 ℃) don fiye da 30min, homogenize kafin amfani.
9.2.2 Samo microwells da ake buƙata kuma mayar da sauran cikin jakar kulle-kulle a 2-8 ℃ nan da nan.
9.2.3 A mayar da hankali bayani na wanke wanke da mayar da hankali cire bayani ya kamata a rewarmed su kasance a dakin zafin jiki kafin amfani.
9.2.4Lamba:An ƙididdige kowane matsayi na microwell kuma duk ƙa'idodi da samfurori yakamata a gudanar da su cikin kwafi.Yi rikodin ma'auni da samfurin matsayi.
9.2.5Dilution na maida hankali maganin antibody: diluted da maida hankali bayani enzyme tare da enzyme conjugate diluents a cikin girma rabo na 1:10 (1 fold mayar da hankali enzyme bayani: 10 folds enzyme conjugate diluents).
9.2.6Ƙara daidaitaccen bayani/samfuri da maganin conjugate enzyme: ƙara 50µl na daidaitaccen bayani ko shirya samfurin zuwa rijiyoyin da suka dace, ƙara 50µl enzyme conjugate bayani.Mix a hankali ta hanyar girgiza farantin da hannu kuma a sanya shi tsawon minti 30 a 25 ℃ tare da murfin.
9.2.7Wanka: Cire murfin a hankali kuma a zubar da ruwa daga cikin rijiyoyin kuma kurkura microwells tare da 250µl diluted wanke bayani (mafita6) a tazara na 10s na 4-5 sau.Shaye ragowar ruwan da takarda mai shayarwa (sauran kumfa na iska za a iya kawar da shi tare da tip mara amfani).
9.2.8Launi: Ƙara 50µl substrate bayani A da 50ul substrate bayani B zuwa kowace rijiya.Mix a hankali ta hanyar girgiza farantin da hannu kuma a sanya shi na 15min a 25 ℃ tare da murfin (duba 12.8).
9.2.11Auna: Ƙara 50µl maganin tasha zuwa kowace rijiya.Mix a hankali ta hanyar girgiza farantin da hannu kuma auna abin sha a 450nm (Ya ba da shawarar auna tare da tsayin dual-wavelength na 450/630nm. Karanta sakamakon a cikin 5min bayan ƙarin bayani tasha.)
10 Sakamako
10.1Yawan shan kashi
Ma'anar ma'auni na ƙimar ƙimar da aka samu don ma'auni da samfurori an raba su ta hanyar ƙimar ƙimar ma'aunin farko (misali sifili) kuma an ninka ta 100%.Ma'aunin sifili don haka ana yin daidai da 100% kuma ana ƙididdige ƙimar sha cikin kashi dari.
=
B ——ma'aunin sha (ko samfurin)
B0 ——shar da sifili misali
10.2Standard Curve
----Don zana daidaitaccen lankwasa: Ɗauki ƙimar ma'auni azaman y-axis, Semi logarithmic na maida hankali na daidaitaccen bayani na AOZ (ppb) azaman x-axis.
---- Matsakaicin AOZ na kowane samfurin (ppb), wanda za'a iya karantawa daga ma'auni na calibration, an ninka ta hanyar ma'auni mai mahimmanci na kowane samfurin da aka biyo baya, kuma ana samun ainihin samfurin samfurin.
Da fatan za a lura:
An ƙirƙira software na musamman don duk raguwar bayanai, waɗanda za'a iya bayarwa akan buƙata.
Fatar dilution…………………………………………………………………………
10.Hankali, daidaito da daidaito
Hankali: 0.025 pb
Iyakar ganowa:
Nama (tsoka, hanta)………………………………………………………. 0.1ppb
Zuma ------------------------------------------------- - 0.1 pb
Daidaito:
Naman dabba (tsokoki da hanta) …………………………………………………………… 100± 20%
zuma………………………………………………………………………………………………………………
Daidaito:CV na kit ɗin ELISA bai wuce 10% ba.
11.Sanarwa
12.1 Ma'anar ƙimar ƙimar ƙimar da aka samu don ma'auni kuma samfuran za a rage su idan ba a tsara reagents da samfuran zuwa zafin jiki ba (20-25 ℃).
12.2 Kada ka ƙyale microwells su bushe tsakanin matakai don kauce wa sake haifar da rashin nasara da kuma aiki mataki na gaba nan da nan bayan danna mariƙin microwells.
12.3 Girgiza kowane reagent a hankali kafin amfani.
12.4 Ka nisantar da fata daga maganin tasha domin ita ce 0.5MH2SO4mafita.
12.5 Kada ku yi amfani da kayan aikin da suka wuce.Kada a musanya reagents na batches daban-daban, in ba haka ba zai sauke hankali.
12.6 Yanayin ajiya: Rike kayan ELISA a 2-8 ℃, kar a daskare.Rufe faranti na microwell, guje wa hasken rana kai tsaye yayin duk abubuwan da suka faru.Ana ba da shawarar rufe faranti na microtiter.
12.7 Ya kamata a watsar da maganin substrate idan ya juya launuka.Reagents na iya zama lalacewa idan ƙimar karɓar (450/630nm) na ma'aunin sifili bai wuce 0.5 (A450nm <0.5).
12.8 Halin launi yana buƙatar 15min bayan ƙarin bayani na substrate;Kuna iya tsawaita lokacin shiryawa zuwa 20min ko fiye idan launi ya yi haske sosai don tantancewa., Kada ku wuce 25min. Akasin haka, rage lokacin shiryawa yadda ya kamata.
12.9 Mafi kyawun zafin jiki shine 25 ℃.Mafi girma ko ƙananan zafin jiki zai haifar da canje-canje na hankali da ƙimar sha.
12.Yanayin ajiya da lokacin ajiya
Yanayin ajiya: 2-8 ℃.
Lokacin ajiya: watanni 12.
