imveliso

1. 1.Imvelaphi

I-Nitrofurans zi-antibiotics zokwenziwa ezibanzi, ezihlala zisetyenziswa kwimveliso yezilwanyana ngenxa yeempawu zayo ezibalaseleyo zokulwa ne-antibacterial kunye ne-pharmacokinetic.Zaziphinde zasetyenziswa njengabakhuthazi bokukhula kwihagu, iinkukhu kunye nemveliso yasemanzini.Kwizifundo zexesha elide kunye nezilwanyana zaselebhu zibonise ukuba amachiza omzali kunye ne-metabolites yawo abonise iimpawu ze-carcinogenic kunye ne-mutagenic.Oku kuye kwakhokelela ekuthintelweni kwe-nitrofurans kunyango lwezilwanyana ezisetyenziselwa ukuveliswa kokutya.Izidakamizwa ze-nitrofuran i-furaltadone, i-nitrofurantoin kunye ne-nitrofurazone zavalwa ukusetyenziswa kwimveliso yezilwanyana zokutya kwi-EU kwi-1993, kwaye ukusetyenziswa kwe-furazolidone kwakungavunyelwe kwi-1995.

Uhlalutyo lwentsalela ye-nitrofuran yamachiza kufuneka lusekelwe ekufumaneni i-metabolites ebophelelwe kwizicubu zamachiza omzali we-nitrofuran.Ekubeni iziyobisi zabazali zixutywe ngokukhawuleza, kwaye i-tissue eboshiwe i-nitrofuran metabolites iya kugcina ixesha elide, ezi metabolites zisetyenziswa njengento ekujoliswe kuyo ekubonweni kokusetyenziswa kakubi kwe-nitrofurans, equka i-Furazolidone metabolite (AOZ), i-Furaltadone metabolite (AMOZ). ), i-Nitrofurantoin metabolite (AHD) kunye ne-Nitrofurazone metabolite (SEM).

Iintsalela ze-AOZ zichongwa ngokuqhelekileyo yi-LC-MS okanye i-LC-MS/MS.Uvavanyo lwe-Enzyme immunoassays, xa luthelekiswa neendlela zechromatographic, lubonisa inzuzo enkulu malunga nobuntununtunu, umda wokubona, izixhobo zobugcisa kunye nexesha elifunekayo.(Iindleko zexesha: 45min)

1. 2.Umgaqo woVavanyo

Le khithi ye-ELISA yenzelwe ukufumanisa i-AOZ ngokusekelwe kumgaqo-nkqubo we-enzyme immunoassay engathanga ngqo.Imithombo ye-microtiter ifakwe kwi-antigen edibeneyo ye-BSA.I-AOZ kwisampulu ikhuphisana ne-antigen egqunywe kwipleyiti ye-microtiter ye-antibody eyongeziweyo.Emva kokongezwa kwe-enzyme conjugate, i-substrate ye-chromogenic isetyenziswa kwaye umqondiso ulinganiswa nge-spectrophotometer.I-absorption ihambelana ngokungafaniyo ne-AOZ yoxinaniso kwisampulu.

1. 3.Usetyenziso

Le khithi ingasetyenziselwa uhlalutyo lobungakanani kunye nomgangatho wentsalela ye-AOZ kwiiithishu ze-anima(izihlunu, isibindi njalo njalo), ubusi.

1. 4.Iimpendulo ezinqamlezayo

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%

INitrofurantoin…………………………………………….…<1%

INitrofurazone…………………………………………..…<1%

1. 5.Izinto Eziyimfuneko

5.1Izixhobo

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

----Umphunga ojikelezayo okanye izixhobo zokomisa initrogen

----Homogenizer /stomacher

-----Shaker

---- Umxube weVortex

-----Iziko

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

----Uthweswe isidanga: 10ml

----Ibhalbhu yerabha yepayipi yerabha

-----Iflaski yeVolumetric: 100ml

-----Iflaski yeglasi: 10ml

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

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

250ul-multipipette

5.2Iireagents

----Ethyl acetate (AR)

----n-hexane (okanye n-heptane) (AR)

----Dipotassium hydrogen phosphate trihydrate

(K₂HPO₄.3H₂O) (AR)

----I-Hydrochloric acid egxininisiweyo (HCl, AR)

-----Methanol

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

---- Amanzi agayiweyo

1. 6.Izixhobo zeKit

l Ipleyiti yeMicrotiter eqatywe nge-antigen, amaqula angama-96

l Izisombululo ezisemgangathweni (iibhotile ezi-6, 1ml/ibhotile)

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

l Ulawulo olusemgangathweni lwe-Spiking : (1ml/ibhotile).....….100ppb

l I-enzyme egxininisiweyo idibanisa i-1ml........i-cap ebomvu

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

l I-Substrate A 7ml………………………..…..ikepusi emhlophe

l I Substrate B 7ml………………………………………..ikepusi ebomvu

l Stop solution 7ml ………………………………ikepusi etyheli

l 20 × isisombululo sokuhlamba esigxininisiweyo 40ml

…………………………………………………icap ecacileyo

l 2×isisombululo esigxininisiweyo sokutsalwa 60ml….ikepusi eluhlaza okwesibhakabhaka

l 2-Nitrobenzaldehyde 15.1mg………………ikepusi emhlophe

1. 7.Ukulungiswa kweeReagents

Isisombululo 1: i-reagent ephuma kuyo:

Yongeza i-10ml yemethanol kwibhotile ene-2-Nitrobenzaldehyde kwaye unyibilike.(kugxininiso lwe-10mM).

Isisombululo 2: 0.1MK₂HPO₄isisombululo:

Ubunzima 22.8g K₂HPO₄.3H₂O ukuya kwi-1L yamanzi adiyiniweyo ukuze anyibilike.

Isisombululo 3: 1M isisombululo seHCl

Dissolve 8.3ml Concentrated hydrochloric acid ngamanzi adiyoni ukuya kwi-100ml.

Isisombululo 4:Isisombululo se-1M NaOH

Chitha i-4g ye-NaOH nge-100ml yamanzi adibeneyo.

Isisombululo 5: isisombululo sokukhupha:

Nciphisa i-2 × isisombululo se-excentrated extraction kunye namanzi adibeneyo kwi-volume ratio ye-1: 1.Esi sisombululo sinokugcinwa i-1month kwi-4 ℃, eya kusetyenziselwa ukukhutshwa kweesampuli.

Isisombululo 6: isisombululo sokuhlamba:

Nciphisa i-20 × isisombululo sokuhlamba esigxininisiweyo ngamanzi adibeneyo kwi-volume ration ye-1:19, eya kusetyenziselwa ukuhlamba iiplate.Esi sisombululo sihlanjululweyo sinokugcinwa inyanga enye kwi-4℃.

1. 8.Amalungiselelo Isampulu

8.1Isaziso kunye nezilumkiso phambi kokusebenza:

a) Nceda usebenzise iingcebiso zolunye kuvavanyo, kwaye utshintshe iingcebiso xa ufunxa i-reagent eyahlukileyo.

b) Qinisekisa ukuba zonke izixhobo zokulinga zicocekile.

c) i-reagent ephuma kuyo inokugcinwa kwi-2-8℃ kangangeenyanga ezintathu;

d) Isisombululo seHCl sinokugcinwa kwiqondo lobushushu begumbi kangangeenyanga ezi-3;

e) Isisombululo se-NaOH sinokugcinwa kwiinyanga ezi-3 kwiqondo lokushisa;

f) Gcina iisampulu ezingaphathwanga kumkhenkce (-20℃);

g) Iisampulu ezinyangweyo zinokugcinelwa i-24h kwi-2-8℃ ebumnyameni.

8.2 Izicubu zezilwanyana kunye neesampuli zesibindi:

-----Homogenize iisampulu nge-homogenizer;

----Ubunzima 1.0±0.05g besampulu yethishu ehomogenized kwityhubhu ye-polystyrene centrifuge eyi-50ml.Yongeza i-4ml yamanzi adibeneyo, i-0.5ml 1M isisombululo se-HCl kunye ne-100ul derivative reagent (jonga isisombululo 1).Yivuthulule i-2min.

---- Fukamisa ngo-37℃ ngobusuku ( malunga ne-16h);

---- Yongeza i-5ml 0.1MK₂HPO₄ (isisombululo2), 0.4ml 1M NaOH (isisombululo4) kunye ne-5ml ethyl acetate.Shake ngokukrakra kwi-30s;

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

---- Thatha i-2.5ml yesigaba se-organic supernatant kwityhubhu yeglasi ecocekileyo eyi-10ml, yome nge-50-60℃ yegesi yenitrogen okanye i-evaporator ejikelezayo;

---- Nyibilikisa intsalela eyomileyo nge-1ml n-hexane (okanye i-n- heptane), i-vortex ye-30s, yongeza isisombululo se-1ml sokukhupha (isisombululo5), vortex 1min, xuba ngokupheleleyo.

---- I-Centrifuge kwiqondo lobushushu begumbi (20-25^oC) i-5min, ubuncinane i-3000g;

---- Susa isigaba se-organic supernatant;thatha i-50μl yesigaba samanzi angaphantsi ukuze uvavanye.

8.4 Ubusi

----nzima 1.0±0.05g yesampulu yobusi obuyi-homogenized kwityhubhu ye-polystyrene centrifuge eyi-50ml;

----Yongeza 4ml amanzi adiyoni, 0.5ml 1M HCl (isisombululo3) kunye ne-100μl ephuma kwi-reagent (isisombululo1);xubha ngokupheleleyo i-2min;

----Fumana nge-37℃ ngobusuku (malunga ne-16h);

----Yongeza i-5ml 0.1MK₂HPO₄ (isisombululo2), 0.4ml 1M NaOH (isisombululo4) kunye ne-5ml ethyl acetate, xubha ngokukrakra kwi-30s;

----I-Centrifuge kwiqondo lobushushu begumbi (20-25℃) i-10min, ubuncinane i-3000g;

----Thatha i-2.5ml yesigaba se-organic supernatant kwityhubhu yeglasi ecocekileyo eyi-10ml, yome nge-50-60℃ igesi yenitrogen okanye i-evaporator ejikelezayo;

----Dibanisa intsalela eyomileyo nge-1ml n-hexane (okanye i-n- heptane), i-vortex ye-30s, yongeza isisombululo se-1ml sokukhupha (isisombululo5), vortex 1min, xuba ngokupheleleyo.

----I-Centrifuge kwiqondo lobushushu begumbi (20-25^oC) i-10min, ubuncinane i-3000g;

----Susa inqanaba le-organic elinamandla;thatha i-50μl yesigaba samanzi angaphantsi ukuze uvavanye.

1. 9.Inkqubo yovavanyo

9.1Qaphela ngaphambi kovavanyo

9.1.1 Qinisekisa ukuba zonke ii-reagents kunye ne-microwells zonke zikwiqondo lobushushu legumbi (20-25℃).

9.1.2 Buyisela zonke ezinye ii-reagents ku-2-8℃ ngoko nangoko emva kokusetyenziswa.

9.1.3 Ukuhlamba iimicrowells ngokuchanekileyo linyathelo elibalulekileyo kwinkqubo yovavanyo;yeyona nto ibalulekileyo ekuveliseni kwakhona uhlalutyo lwe-ELISA.

9.1.4 Kuphephe ukukhanya kwaye wogqume iimicrowells ngexesha lokufukamela.

9.2 Amanyathelo oVavanyo

9.2.1 Thatha zonke ii-reagents kwiqondo lobushushu legumbi (20-25℃) ngaphezu kwe-30min, i-homogenize phambi kokusetyenziswa.

9.2.2 Khupha ii-microwells ezifunekayo kwaye ezinye uzibuyisele kwi-zip-lock bag ku-2-8℃ ngokukhawuleza.

9.2.3 Isisombululo sokuhlamba esigxininisiweyo kunye nesisombululo esigxininisiweyo sokukhupha kufuneka siphinde sifudunyezwe ukuze sibe kwiqondo lokushisa ngaphambi kokusetyenziswa.

9.2.4Inani:Inani lezithuba zemicrowell zonke kunye nemigangatho kunye neesampulu kufuneka ziqhutywe ngokuphindwe kabini.Rekhoda imigangatho kunye neendawo zeesampulu.

9.2.5Unyibiliko lwesisombululo esigxininisiweyo se-antibody: nciphisa isisombululo se-enzyme egxininisiweyo kunye ne-enzyme ye-conjugate diluents kwi-volume ratio ye-1: 10 (i-1 fold concentrated enzyme solution: i-10 folds enzyme conjugate diluents).

9.2.6Yongeza isisombululo esisemgangathweni / isampuli kunye nesisombululo se-enzyme conjugate: yongeza i-50µl yesisombululo esiqhelekileyo okanye isampuli esilungisiweyo kumaqula ahambelanayo, yongeza i-50µl isisombululo se-enzyme conjugate.Xuba ngobunono ngokushukumisa ipleyiti ngesandla kwaye ufukame i-30min kwi-25℃ ngegquma.

9.2.7Hlamba: Susa isigqubuthelo ngobunono kwaye ugalele ulwelo oluphuma equleni kwaye uhlambulule iimicrowells nge 250µl isisombululo sokuhlamba esixutyiweyo (isisombululo6) ngexesha le-10 ngamaxesha angama-4-5.Funxa amanzi ashiyekileyo ngephepha elifunxayo (iqamza lomoya eliseleyo linokupheliswa ngencam engasetyenziswanga).

9.2.8Umbala: Yongeza i-50µl substrate isisombululo A kunye ne-50ul substrate isisombululo B kwiqula ngalinye.Xuba ngobunono ngokushukumisa ipleyiti ngesandla kwaye ufukame i-15min kwi-25℃ ngegquma (bona 12.8).

9.2.11Umlinganiselo: Yongeza 50µl isisombululo sokumisa kwiqula ngalinye.Xuba ngobunono ngokugungqisa ipleyiti ngesandla kwaye ulinganise ukufunxa kwi-450nm (Icetyisiwe ukulinganisa nge-double-wavelength ye-450/630nm. Funda umphumo ngaphakathi kwe-5min emva kokongezwa kwesisombululo sokumisa.)

10 Iziphumo

10.1Ipesenti yokuthatha

Amaxabiso aphakathi amaxabiso e-absorbence afunyenwe kwimigangatho kunye neesampuli zahlulwe ngexabiso lokuthatha umgangatho wokuqala (umgangatho we-zero) kwaye zanda nge-100%.Umgangatho we-zero wenziwa ulingane ne-100% kwaye amaxabiso e-absorpsor acatshulwe ngokweepesenti.

=

B ——umgangatho wokungabikho (okanye isampuli)

B0 —-absorbance zero umgangatho

10.2Ijika eliMgangatho

----Ukuzoba igophe elisemgangathweni: Thatha ixabiso lokufunxa lemigangatho njenge-y-axis, isemi logarithmic yoxinaniso lwesisombululo esisezantsi se-AOZ (ppb) njenge-x-axis.

----I-AOZ i-concentration yesampuli nganye (ppb), enokufundwa kwi-curve yokulinganisa, iphindwe nge-dilution factor ehambelanayo yesampuli nganye elandelwayo, kwaye i-concentration yangempela yesampuli ifunyenwe.

Nceda uqaphele:

Isoftware ekhethekileyo iye yaphuhliswa kuko konke ukuncitshiswa kwedatha, enokunikezelwa ngesicelo.

Dilution factor………………………………………………………2

10.Uvakalelo, ukuchaneka kunye nokuchaneka

Uvakalelo: 0.025ppb

Umda wokufunyanwa:

Izicubu (izihlunu, isibindi)…………………………………

Ubusi------------------------------------------------- -0.1ppb

Ukuchaneka:

Izicubu zezilwanyana (izihlunu nesibindi)……………………100±20%

Ubusi………………………………………………….. 100±20%

Ukuchaneka:I-CV yekhithi ye-ELISA ingaphantsi kwe-10%.

11.Qaphela

12.1 Amaxabiso aphakathi kwamaxabiso afunyenwe kwimigangatho kunye neesampuli ziya kuncitshiswa ukuba i-reagents kunye neesampuli azizange zilawulwe kwiqondo lokushisa (20-25℃).

12.2 Musa ukuvumela ii-microwells ukuba zome phakathi kwamanyathelo ukuphepha ukuphindaphinda okungaphumelelanga kwaye usebenzise inyathelo elilandelayo ngokukhawuleza emva kokucofa isibambi se-microwells.

12.3 Vuthulula i-reagent nganye ngobunono phambi kokusetyenziswa.

12.4 Gcina ulusu lwakho kude nesisombululo sokumisa kuba yi-0.5MH₂SO₄isisombululo.

12.5 Musa ukusebenzisa iikhithi eziphelelwe lixesha.Musa ukutshintshisa ii-reagents zeebhetshi ezahlukeneyo, okanye iya kuwisa ubuntununtunu.

12.6 Imeko yogcino: Gcina iikhithi ze-ELISA ku-2-8℃, musa ukuba ngumkhenkce.Zitywine iipleyiti ze-microwell zokuphumla, Kuphephe ukukhanya kwelanga ngexesha lonke lokufukamela.Kunconywa ukugubungela iiplati ze-microtiter.

12.7 Isisombululo seSubstrate kufuneka siyekwe ukuba sijika imibala.I-reagents inokuthi ihlaziywe ukuba ixabiso lokuxhamla (450 / 630nm) lomgangatho we-zero lingaphantsi kwe-0.5 (A450nm <0.5).

12.8 Ukusabela kombala kufuna i-15min emva kokongezwa kwesisombululo se-substrate;Unokwandisa ixesha lokufukamela ukuya kwi-20min okanye ngaphezulu ukuba umbala ulula kakhulu ukuba ungamiselwa., ungaze udlule i-25min. Ngokuchasene noko, nciphisa ixesha lokufukamela ngokufanelekileyo.

12.9 Elona qondo lobushushu lokusabela liphezulu ngama-25℃.Ubushushu obuphezulu okanye obuphantsi buya kukhokelela ekutshintsheni kobuntununtunu kunye nexabiso lokuthatha.

12.Imeko yokugcina kunye nexesha lokugcina

Imeko yokugcina: 2-8℃.

Ixesha lokugcinwa: 12months.