umkhiqizo

1. 1.Ingemuva

I-Nitrofuran iyimithi elwa namagciwane yokwenziwa ebanzi, evame ukusetshenziswa ekukhiqizeni izilwane ngenxa yezakhiwo zayo ezinhle kakhulu zokulwa namagciwane kanye ne-pharmacokinetic.Ayephinde asetshenziswa njengabagqugquzeli bokukhula kwengulube, izinkukhu kanye nokukhiqizwa kwamanzi.Ezifundweni zesikhathi eside ngezilwane zaselabhu zabonisa ukuthi izidakamizwa zomzali kanye nama-metabolite azo akhombisa izici ze-carcinogenic kanye ne-mutagenic.Lokhu sekuholele ekuvinjweni kwe-nitrofuran ekwelapheni izilwane ezisetshenziselwa ukukhiqiza ukudla.Izidakamizwa ze-nitrofuran i-furaltadone, i-nitrofurantoin ne-nitrofurazone zavinjelwa ukusetshenziswa ekukhiqizeni izilwane zokudla e-EU ngo-1993, futhi ukusetshenziswa kwe-furazolidone kwakuvinjelwe ku-1995.

Ukuhlaziywa kwezinsalela zemithi ye-nitrofuran kufanele kusekelwe ekutholweni kwe-metabolites eboshwe ezicutshini zemithi engumzali we-nitrofuran.Njengoba izidakamizwa zabazali zenziwa ngokushesha okukhulu, futhi izicubu eziboshwe i-nitrofuran metabolites zizogcinwa isikhathi eside, lawa ma-metabolites asetshenziswa njengento ehlosiwe ekutholeni ukuhlukunyezwa kwe-nitrofurans, okuhlanganisa i-Furazolidone metabolite (AOZ), i-Furaltadone metabolite (AMOZ). ), i-Nitrofurantoin metabolite (AHD) kanye ne-Nitrofurazone metabolite (SEM).

Izinsalela ze-AOZ zinqunywa kakhulu yi-LC-MS noma i-LC-MS/MS.Ama-Enzyme immunoassay, uma kuqhathaniswa nezindlela zechromatographic, akhombisa izinzuzo ezinkulu mayelana nokuzwela, umkhawulo wokutholwa, imishini yobuchwepheshe kanye nesidingo sesikhathi.(Izindleko zesikhathi: 45min)

1. 2.Isimiso Sokuhlola

Le khithi ye-ELISA yakhelwe ukuthola i-AOZ ngokususelwe kumgomo we-enzyme immunoassay engaqondile yokuncintisana.Imithombo ye-microtiter imbozwe nge-antigen exhumene ne-BSA.I-AOZ kusampula iqhudelana ne-antigen embozwe epuleti le-microtiter ye-antibody engeziwe.Ngemuva kokwengezwa kwe-enzyme conjugate, i-substrate ye-chromogenic isetshenziswa futhi isignali ikalwa nge-spectrophotometer.Ukumuncwa kuhambisana ngokuphambene nokugxilisana kwe-AOZ kusampula.

1. 3.Izinhlelo zokusebenza

Le khithi ingasetshenziswa ekuhlaziyweni komthamo kanye nekhwalithi yensalela ye-AOZ ngaphakathiizicubu ze-anima(imisipha, isibindi njll), uju.

1. 4.Ukusabela okuphambene

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

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

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

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

I-Furazolidone……………………………………………..… 16.3%

Furaltadone……………………………………………….…<1%

I-Nitrofurantoin…………………………………………….…<1%

I-Nitrofurazone…………………………………………..…<1%

1. 5.Izinto Ezidingekayo

5.1Izisetshenziswa

---- I-Microtiter plate spectrophotometer (450nm/630nm)

----I-Rotary evaporator noma amathuluzi okomisa i-nitrogen

----Homogenizer /stomacher

-----Shaker

---- I-Vortex mixer

---- I-Centrifuge

----Ibhalansi yokuhlaziya (i-inductance: 0.01g)

---- I-pipette ethweswe iziqu: 10ml

---- I-rubber pipette bulb

-----I-Volumetric flask: 100ml

---- Iflask yengilazi: 10ml

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

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

250ul-multipipette

5.2Ama-reagents

----I-Ethyl acetate (AR)

----n-hexane (noma i-n-heptane) (AR)

---- I-Dipotassium hydrogen phosphate trihydrate

(K₂I-HPO₄.3H₂O) (AR)

----I-hydrochloric acid egxilile (HCl, AR)

-----I-Methanol

----I-sodium hydroxide (NaOH, AR)

---- Amanzi akhishiwe

1. 6.Izingxenye Zekhithi

l Ipuleti leMicrotiter elihlanganiswe ne-antigen, imithombo engama-96

l Izixazululo ezijwayelekile (amabhodlela ayi-6, 1ml/ibhodlela)

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

l Ukulawula okujwayelekile kwe-spiking : (1ml/ibhodlela)........100ppb

l I-enzyme egxilile ihlanganisa i-1ml........ikhephu ebomvu

l I-enzyme conjugate diluents 10ml ………..ikepisi eliluhlaza

l I-Substrate A 7ml………………………..…..ikepisi elimhlophe

l I-Substrate B 7ml………………………………….…..ikepisi elibomvu

l Isixazululo sokumisa 7ml………………………………ikepisi eliphuzi

l 20 × isixazululo esigxilile sokugeza 40ml

………………………………………………ikhephu esobala

l 2×isixazululo esigxilisiwe sokukhipha 60ml….ikepisi eliluhlaza okwesibhakabhaka

l 2-Nitrobenzaldehyde 15.1mg…………………ikepisi elimhlophe

1. 7.Ukulungiswa kwama-reagents

Isixazululo 1: i-reagent ephuma kokunye:

Engeza u-10ml we-methanol ebhodleleni eline-2-Nitrobenzaldehyde bese uhlakazeka.(ekuhlanganiseni kwe-10mM).

Isixazululo 2: 0.1MK₂I-HPO₄Isixazululo:

Isisindo 22.8g K₂I-HPO₄.3H₂O ukuya ku-1L wamanzi akhishiwe azoncibilika.

Isixazululo 3: Isixazululo se-HCl esingu-1M

Chaza 8.3ml Concentrated hydrochloric acid ngamanzi e-deionized kuya ku-100ml.

Isixazululo 4:Isixazululo se-1M NaOH

Chaza i-4g NaOH ngamanzi a-deionized angu-100ml.

Isixazululo 5: isixazululo sokukhipha:

Nciphisa isixazululo se-2 × esigxilisiwe sokukhipha ngamanzi akhishwe ngevolumu ngesilinganiso se-1: 1.Lesi sixazululo singalondolozwa inyanga engu-1 ku-4℃, esizosetshenziselwa ukukhipha amasampula.

Isixazululo 6: isixazululo sokugeza:

Nciphisa isixazululo sokugeza esigxilile esingu-20 × ngamanzi angcolile ngesilinganiso sevolumu engu-1:19, esizosetshenziselwa ukugeza amapuleti.Lesi sixazululo esihlanjululwe singalondolozwa inyanga engu-1 ku-4 ℃.

1. 8.Amalungiselelo Esampula

8.1Izaziso nezixwayiso ngaphambi kokusebenza:

a) Sicela usebenzise amathiphu aphuma kanye esivivinyweni, futhi ushintshe amathiphu lapho udonsa i-reagent ehlukile.

b) Qiniseka ukuthi zonke izinsimbi zokuhlola zihlanzekile.

c) i-reagent ephuma kokunye ingagcinwa ku-2-8℃ izinyanga ezintathu;

d) Isixazululo se-HCl singagcinwa ezingeni lokushisa elilingana negumbi izinyanga ezi-3;

e) Isixazululo se-NaOH singalondolozwa izinyanga ezi-3 ekamelweni lokushisa;

f) Gcina amasampula angakapheki emakhazeni(-20℃);

g) Amasampula alashiwe angagcinelwa amahora angu-24 ku-2-8℃ ebumnyameni.

8.2 Amasampula ezicubu zezilwane nesibindi:

-----Homogenize amasampuli nge-homogenizer;

----Isisindo esingu-1.0±0.05g sesampula yethishu ehlanganisiwe ibe yishubhu ye-polystyrene centrifuge engu-50ml.Engeza amanzi a-deionized angu-4ml, isixazululo esingu-0.5ml 1M HCl kanye ne-reagent derivative engu-100ul (bona isisombululo 1).Yinyakazise imizuzu emi-2.

---- Fukamela ngo-37℃ ngobusuku obubodwa ( cishe ngo-16h);

---- Engeza u-5ml 0.1MK₂I-HPO₄ (isisombululo2), 0.4ml 1M NaOH (isisombululo4) kanye ne-5ml ethyl acetate.Nyakazisa kanzima iminyaka engama-30;

---- I-Centrifuge ekamelweni lokushisa (20-25℃) i-10min, okungenani i-3000g;

---- Thatha u-2.5ml wesigaba sezinto eziphilayo ezingaphezu kuka-10ml eshubhu lengilazi elihlanzekile, lome ngegesi yenitrogen engu-50-60℃ noma i-evaporator ejikelezayo;

---- Chaza insalela eyomile nge-1ml n-hexane (noma i-n- heptane), i-vortex iminyaka engu-30, engeza isixazululo sokukhipha esingu-1ml (isisombululo5), vortex 1min, hlanganisa ngokuphelele.

---- I-Centrifuge ekamelweni lokushisa (20-25^oC) for 5min, okungenani 3000g;

---- Susa isigaba se-organic supernatant;thatha u-50μl wesigaba samanzi esingaphansi ukuze uhlole.

8.4 Uju

----linganisa u-1.0±0.05g wesampula yoju lwe-homogenized lube yishubhu ye-polystyrene centrifuge engu-50ml;

----Engeza amanzi akhiqiziwe angu-4ml, 0.5ml 1M HCl (isisombululo3) kanye ne-reagent ephuma kokunye engu-100μl (isisombululo1);shake ngokuphelele imizuzu emi-2;

----Fukamela ngo-37℃ ngobusuku obubodwa (cishe ngo-16h);

----Engeza u-5ml 0.1MK₂I-HPO₄ (isisombululo2), 0.4ml 1M NaOH (isisombululo4) kanye ne-5ml ethyl acetate, qhaqhazela kakhulu iminyaka engama-30;

----Centrifuge ekamelweni lokushisa (20-25℃) 10min, okungenani 3000g;

----Thatha u-2.5ml wesigaba sezinto eziphilayo ezingaphezu kuka-10ml eshubhu lengilazi elihlanzekile, lome ngegesi yenitrogen engu-50-60℃ noma i-rotary evaporator;

----Chaza insalela eyomile nge-1ml n-hexane (noma i-n- heptane), i-vortex iminyaka engama-30, engeza isixazululo sokukhipha esingu-1ml (isisombululo5), vortex 1min, hlanganisa ngokuphelele.

----Centrifuge ekamelweni lokushisa (20-25^oC) for 10min, okungenani 3000g;

----Susa isigaba sezinto eziphilayo ezingaphezu kwemvelo;thatha u-50μl wesigaba samanzi esingaphansi ukuze uhlole.

1. 9.Inqubo yokuhlola

9.1Qaphela ngaphambi kokuhlolwa

9.1.1 Qinisekisa ukuthi zonke izinto ezisebenza ngogesi nama-microwell konke kusezingeni lokushisa elilingana negumbi (20-25℃).

9.1.2 Buyisela wonke amanye ama-reagents ku-2-8℃ ngokushesha ngemva kokusetshenziswa.

9.1.3 Ukugeza ama-microwell ngendlela efanele kuyisinyathelo esibalulekile ohlelweni lokuhlola;kuyisici esibalulekile ekukhiqizeni kabusha kokuhlaziywa kwe-ELISA.

9.1.4 Gwema ukukhanya futhi umboze ama-microwell ngesikhathi sokufukamela.

9.2 Izinyathelo Zokuhlola

9.2.1 Khipha wonke ama-reagents ekamelweni lokushisa (20-25℃) ngaphezu kwama-30min, i-homogenize ngaphambi kokusetshenziswa.

9.2.2 Khipha ama-microwell adingekayo bese ubuyisela amanye esikhwameni se-zip-lock ku-2-8℃ ngokushesha.

9.2.3 Isixazululo sokugeza okugxilile kanye nekhambi lokudonsa eligxilile kufanele kufudunyezwe kabusha ukuze kugcinwe izinga lokushisa elilingana negumbi ngaphambi kokusetshenziswa.

9.2.4Inombolo:Ifakwe izinombolo kuzo zonke izindawo ze-microwell nawo wonke amazinga namasampuli kufanele asetshenziswe ngokuphindwe kabili.Rekhoda amazinga kanye nezikhundla zamasampuli.

9.2.5Ukwehliswa kwesixazululo esigxilile se-antibody: hlambulula isisombululo se-enzyme egxilile nge-enzyme conjugate diluents ngesilinganiso sevolumu ye-1:10 (isixazululo se-enzyme egxilile esigoqiwe esingu-1: ukugoqa okungu-10 kwe-enzyme conjugate diluent).

9.2.6Engeza isisombululo esijwayelekile / isampula kanye nesisombululo se-enzyme conjugate: engeza u-50µl wesixazululo esijwayelekile noma isampula elilungisiwe emithonjeni ehambisanayo, engeza isixazululo se-50µl se-enzyme conjugate.Hlanganisa ngobumnene ngokunyakazisa ipuleti ngesandla bese ufukamela imizuzu engama-30 ku-25℃ ngekhava.

9.2.7Geza: Khipha ikhava ngobumnene bese uthela uketshezi emithonjeni bese uhlanza ama-microwells ngesisombululo sokugeza esihlanjululwe esingu-250µl (isisombululo6) ngesikhathi sokuphumula se-10 izikhathi ezingu-4-5.Gcoba amanzi asele ngephepha elimuncayo (ibhamuza lomoya elisele lingaqedwa ngethiphu elingasetshenzisiwe).

9.2.8Umbala: Engeza isixazululo se-substrate esingu-50µl A kanye nekhambi le-substrate elingu-50ul B emthonjeni ngamunye.Hlanganisa ngobumnene ngokunyakazisa ipuleti ngesandla bese ufukamela imizuzu eyi-15 ku-25℃ ngesembozo (bona 12.8).

9.2.11Kala: Engeza isixazululo sokumisa esingu-50µl emthonjeni ngamunye.Xuba ngobumnene ngokunyakazisa ipuleti ngesandla bese ukala ukumunca ku-450nm (Kuphakanyiswe isilinganiso esinobude obubili obungu-450/630nm. Funda umphumela phakathi kwemizuzu emi-5 ngemva kokwengeza isixazululo sokumisa.)

10 Imiphumela

10.1Iphesenti lokumunca

Amanani amaphakathi amavelu okumunca atholwe kumazinga kanye namasampuli ahlukaniswa ngevelu yokumunca yezinga lokuqala (izinga elinguziro) futhi liphindwe ngo-100%.Izinga elinguziro lenziwa lalingana no-100% futhi amanani okumunca acashunwe ngamaphesenti.

=

B ——izinga le-absorbance (noma isampula)

B0 ——absorbance zero standard

10.2Ijika Elijwayelekile

----Ukuze udwebe ijika elijwayelekile: Thatha inani lokumunca lamazinga njenge-y-eksisi, i-semi logarithmic yokugxila kwesixazululo esijwayelekile se-AOZ (ppb) njenge-x-eksisi.

----Ukugxiliswa kwe-AOZ kwesampula ngayinye (ppb), engafundwa kwijika lokulinganisa, kuphindwa ngesici esihambisanayo sokuhlanjululwa kwesampula ngayinye elandelwayo, futhi ukugxiliswa kwangempela kwesampula kuyatholakala.

Sicela uqaphele:

Isofthiwe ekhethekile yenzelwe konke ukuncishiswa kwedatha, enganikezwa ngesicelo.

I-Dilution factor……………………………………………………2

10.Ukuzwela, ukunemba nokunemba

Ukuzwela: 0.025ppb

Umkhawulo wokutholwa:

Izicubu (imisipha, isibindi)……………………………….0.1ppb

Uju------------------------------------------------ -0.1ppb

Ukunemba:

Izicubu zezilwane (imisipha nesibindi)……………………100±20%

Uju………………………………………………….. 100±20%

Ukunemba:I-CV yekhithi ye-ELISA ingaphansi kwe-10%.

11.Qaphela

12.1 Amanani amaphakathi wamanani okumunca atholwe kumazinga kanye namasampuli azoncishiswa uma ama-reagents namasampuli engalawulwanga kukushisa kwegumbi (20-25℃).

12.2 Ungavumeli ama-microwells ome phakathi kwezinyathelo ukugwema ukukhiqizwa kabusha okungaphumeleli futhi usebenzise isinyathelo esilandelayo ngokushesha ngemva kokuthepha isibambi sama-microwells.

12.3 Nyakazisa i-reagent ngayinye ngobumnene ngaphambi kokuyisebenzisa.

12.4 Gcina isikhumba sakho kude nesixazululo sokumisa ngoba singu-0.5MH₂SO₄isisombululo.

12.5 Ungawasebenzisi amakhithi asephelelwe yisikhathi.Ungashintshisani ama-reagents amaqoqo ahlukene, kungenjalo kuzokwehlisa ukuzwela.

12.6 Isimo sesitoreji: Gcina amakhithi e-ELISA eku-2-8℃, ungafrizi.Vala amapuleti amancane, Gwema ukukhanya kwelanga okuqondile phakathi nawo wonke ama-incubation.Kunconywa ukumboza amapuleti e-microtiter.

12.7 Isixazululo se-substrate kufanele siyekwe uma sishintsha imibala.Ama-reagents angase ehle uma inani lokumunca (450/630nm) lezinga elinguziro lingaphansi kuka-0.5 (A450nm<0.5).

12.8 Ukusabela kombala kudinga imizuzu engu-15 ngemva kokwengezwa kwesixazululo se-substrate;Ungakwazi ukwandisa isikhathi sokufukamela sibe yi-20min noma ngaphezulu uma umbala ulula kakhulu ukuthi unganqunywa., ungalokothi udlule i-25min. Ngokuphambene nalokho, finyeza isikhathi sokufukamela ngendlela efanele.

12.9 Izinga lokushisa elilungile lokusabela ngu-25℃.Izinga lokushisa eliphakeme noma eliphansi lizoholela ekushintsheni kokuzwela kanye namanani okumunca.

12.Isimo sesitoreji nesikhathi sokugcina

Isimo sesitoreji: 2-8℃.

Isikhathi sokugcina: 12months.