sehlahisoa

1. 1.Ka morao

Li-nitrofuran ke lithibela-mafu tsa maiketsetso tse pharalletseng, tse sebelisoang khafetsa tlhahisong ea liphoofolo bakeng sa thepa ea eona e ntle ea antibacterial le pharmacokinetic.Li ne li boetse li sebelisitsoe e le bahlohlelletsi ba kholo ea likolobe, likhoho le tlhahiso ea metsing.Lithutong tsa nako e telele le liphoofolo tsa laboratori li bonts'itse hore lithethefatsi tsa motsoali le metabolite ea tsona li bonts'a likarolo tsa carcinogenic le mutagenic.Sena se lebisitse ho thibelo ea nitrofuran bakeng sa phekolo ea liphoofolo tse sebelisetsoang tlhahiso ea lijo.Lithethefatsi tsa nitrofuran furaltadone, nitrofurantoin le nitrofurazone li ne li thibetsoe ho sebelisoa tlhahiso ea liphoofolo tsa lijo EU ka 1993, 'me tšebeliso ea furazolidone e thibetsoe ka 1995.

Tlhahlobo ea masala a lithethefatsi a nitrofuran e lokela ho ipapisa le ho sibolloa ha metabolite e tlameletsoeng ke lisele tsa lithethefatsi tsa motsoali oa nitrofuran.Kaha lithethefatsi tsa motsoali li sebelisoa ka potlako haholo, 'me lisele tse tlamang nitrofuran metabolites li tla boloka nako e telele, metabolite tsena li sebelisoa e le sepheo sa ho lemoha tlhekefetso ea nitrofurans, e kenyeletsang Furazolidone metabolite (AOZ), Furaltadone metabolite (AMOZ). ), metabolite ea Nitrofurantoin (AHD) le metabolite ea Nitrofurazone (SEM).

Masala a AOZ a khethoa hangata ke LC-MS kapa LC-MS/MS.Li-immunoassays tsa enzyme, ha li bapisoa le mekhoa ea chromatographic, li bonts'a melemo e mengata mabapi le kutlo, moeli oa ho lemoha, lisebelisoa tsa tekheniki le tlhoko ea nako.(Litšenyehelo tsa nako: 45min)

1. 2.Teko Molao-motheo

Sesebelisoa sena sa ELISA se etselitsoe ho bona AOZ ho ipapisitsoe le molao-motheo oa immunoassay ea enzyme e sa tobang.Liliba tsa microtiter li koahetsoe ka antigen e amanang le BSA.AOZ ka sampole e qothisana lehlokoa le antigen e tlositsoeng holim'a poleiti ea microtiter bakeng sa lesole la 'mele le kentsoeng.Kamora ho eketsoa ha enzyme conjugate, substrate ea chromogenic e sebelisoa mme lets'oao le lekanyetsoa ka spectrophotometer.Ho monya ho ipapisitse le maemo a AOZ sampole.

1. 3.Lisebelisoa

Sesebelisoa sena se ka sebelisoa tlhahlobong ea bongata le ea boleng ba masala a AOZ holithane tsa anima(mesifa, sebete joalo-joalo), mahe a linotsi.

1. 4.Liphetoho tse fapaneng

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

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

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

Nitrofurazone metabolite (SEM)……………………….<0.1%

Furazolidone……………………………………………..… 16.3%

Furaltadone……………………………………………….…<1%

Nitrofurantoin …………………………………………….…<1%

Nitrofurazone……………………………………………..…<1%

1. 5.Lintho Tse Hlokehang

5.1Thepa

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

----Sesebediswa sa rotary evaporator kapa nitrogen drying

----Homogenizer /stomacher

---- Shaker

---- Motsoako oa Vortex

-----Setsi

----Analytical balance (inductance: 0.01g)

-----Pipette e fumaneng mangolo: 10ml

----Lebone la peipi ea rabara

---- Botlolo ea Volumetric: 100ml

---- Botlolo ea khalase: 10ml

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

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

250ul-multipipette

5.2Li-reagents

---- Ethyl acetate (AR)

----n-hexane (kapa n-heptane) (AR)

---- Dipotassium hydrogen phosphate trihydrate

(K₂HPO₄.3H₂O) (AR)

----- Concentrated hydrochloric acid (HCl, AR)

-----Methanol

----Sodium hydroxide (NaOH, AR)

-----Metsi a hloekisitsoeng

1. 6.Likarolo tsa Kit

l Letlapa la Microtiter le koahetsoeng ka antigen, liliba tse 96

l Litharollo tse tloaelehileng (libotlolo tse 6, 1ml/botlolo)

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

l Taolo e tloaelehileng ea Spiking : (1ml/botlolo).....….100ppb

l Enzyme e tsitsitseng e kopanya 1ml........kepisi e khubelu

l Enzyme conjugate diluents 10ml ………..sekoahelo se setala

l Substrate A 7ml ………………………..…..kepisi e tšoeu

l Substrate B 7ml…………………………………….…..kepisi e khubelu

l Stop solution 7ml …………………………………kepisi e mosehla

l 20 × tharollo e tebileng ea ho hlatsoa 40ml

………………………………………………….. transparent cap

l 2 × concentrated extraction solution 60ml….blue cap

l 2-Nitrobenzaldehyde 15.1mg ………………kepisi e tšoeu

1. 7.Ho lokisoa ha li-reagents

Tharollo 1: e tsoang ho reagent:

Kenya 10ml ea methanol botlolong e nang le 2-Nitrobenzaldehyde 'me e qhibilihe.(ka khatello ea 10mM).

Tharollo 2: 0.1MK₂HPO₄tharollo:

Boima ba 22.8g K₂HPO₄.3H₂Ho isa ho 1L ea metsi a deionized ho qhibiliha.

Tharollo 3: 1M HCl tharollo

Qhala 8.3ml Concentrated hydrochloric acid ka metsi a deionized ho 100ml.

Tharollo 4:Tharollo ea 1M NaOH

Hlakola 4g NaOH ka 100ml ea metsi a deionized.

Tharollo 5: tharollo ea ho ntša:

Hlakola tharollo ea 2 × concentrated extraction ka metsi a deionized ka tekanyo ea 1: 1.Tharollo ena e ka bolokoa bakeng sa 1month ho 4 ℃, e tla sebelisoa ho ntša lisampole.

Tharollo 6: tharollo ea ho hlatsoa:

Hlatsoa tharollo ea 20 × concentrated ea ho hlatsoa ka metsi a hloekisitsoeng ka tekanyo ea 1:19, e tla sebelisoa ho hlatsoa lipoleiti.Tharollo ena e hlapollotsoeng e ka bolokoa khoeli e le 1 ho 4℃.

1. 8.Litokisetso tsa Mehlala

8.1Tlhokomeliso le litemoso pele ho ts'ebetso:

a) Ka kopo sebelisa malebela a nako e le 'ngoe tekong, 'me u fetole malebela ha u monya reagent e fapaneng.

b) Etsa bonnete ba hore lisebelisoa tsohle tsa liteko li hloekile.

c) reagent e tsoang ho eona e ka bolokoa ho 2-8 ℃ ka likhoeli tse tharo;

d) Tharollo ea HCl e ka bolokoa mochesong oa kamore bakeng sa likhoeli tse 3;

e) Tharollo ea NaOH e ka bolokoa ka likhoeli tse 3 mocheso oa kamore;

f) Boloka lisampole tse sa sebetsoang ka har'a leqhoa (-20 ℃);

g) Mehlala e phekotsoeng e ka bolokoa bakeng sa 24h ho 2-8 ℃ lefifing.

8.2 Lisele tsa liphoofolo le lisampole tsa sebete:

---- Homogenize disampole ka homogenizer;

---- Boima ba 1.0±0.05g ba sampole ea linama tse homogenized ho 50ml polystyrene centrifuge tube.Eketsa 4ml deionized water, 0.5ml 1M HCl solution le 100ul derivative reagent (sheba tharollo 1).E sisinyehe bakeng sa 2min.

Emela ka 37℃ bosiu bo le bong (hoo e ka bang 16h);

---- Eketsa 5ml 0.1MK₂HPO₄ (tharollo2), 0.4ml 1M NaOH (tharollo4) le 5ml ethyl acetate.Shake ka matla bakeng sa 30s;

---- Centrifuge mocheso oa kamore (20-25℃) bakeng sa 10min, bonyane 3000g;

---- Nka 2.5ml ea mokhahlelo o matlahali oa organic ka har'a tube ea khalase e hloekileng ea 10ml, e omeletseng ka khase ea nitrogen ea 50-60℃ kapa mouoane oa rotary;

---- Hlakola masala a omileng ka 1ml n-hexane (kapa n- heptane), vortex bakeng sa 30s, eketsa 1ml ea thapo ea tharollo (tharollo5), vortex 1min, kopanya ka ho feletseng.

---- Centrifuge mocheso oa kamore (20-25^oC) bakeng sa 5min, bonyane 3000g;

---- Tlosa karolo ea supernatant organic;nka 50μl ea karolo ea metsi a substrate bakeng sa tlhahlobo.

8.4 Mahe a linotsi

---- bekha 1.0±0.05g ea sampole ea mahe a linotsi a homogenized ka har'a 50ml polystyrene centrifuge tube;

----Eketsa 4ml metsi a entsoeng ka deionized, 0.5ml 1M HCl (tharollo3) le 100μl derivative reagent (tharollo1);sisinya ka ho feletseng bakeng sa 2min;

Emela ka 37℃ bosiu bo le bong (hoo e ka bang 16h);

----Eketsa 5ml 0.1MK₂HPO₄ (tharollo2), 0.4ml 1M NaOH (tharollo4) le 5ml ethyl acetate, sisinya ka matla bakeng sa 30s;

----Centrifuge mocheso oa kamore (20-25℃) bakeng sa 10min, bonyane 3000g;

----Nka 2.5ml ea mokhahlelo o matla haholo oa organic o e kenya ka har'a tube ea khalase e hloekileng ea 10ml, e omeletseng ka khase ea nitrogen ea 50-60℃ kapa mouoane oa rotary;

----Qholosa se setseng se omeletseng ka 1ml n-hexane (kapa n- heptane), vortex bakeng sa 30s, eketsa 1ml ea mokelikeli (tharollo5), vortex 1min, kopanya ka ho feletseng.

---- Centrifuge mocheso oa kamore (20-25^oC) bakeng sa 10min, bonyane 3000g;

----Tlosa karolo ea tlhaho e matlahali;nka 50μl ea karolo ea metsi a substrate bakeng sa tlhahlobo.

1. 9.Mokhoa oa tlhahlobo

9.1Ela hloko pele ho tlhahlobo

9.1.1 Etsa bonnete ba hore li-reagents le li-microwell kaofela li mocheso oa kamore (20-25℃).

9.1.2 Khutlisetsa li-reagents tsohle ho 2-8℃ hang ka mor'a ho sebelisoa.

9.1.3 Ho hlatsoa li-microwell ka nepo ke mohato oa bohlokoa tšebetsong ea tlhahlobo;ke ntlha ea bohlokoa ho ikatisa ha tlhahlobo ea ELISA.

9.1.4 Qoba lebone 'me u koahele li-microwell nakong ea ho incubation.

9.2 Mehato ea Tekolo

9.2.1 Ntša li-reagents tsohle ka mocheso oa kamore (20-25℃) ho feta 30min, homogenize pele u sebelisoa.

9.2.2 Ntša li-microwell tse hlokahalang 'me u khutlisetse tse ling ka mokotleng oa zip-lock ka 2-8℃ hang-hang.

9.2.3 Tharollo ea ho hlatsoa e teteaneng le tharollo e tsitsitseng ea ho ntša metsi e lokela ho futhumatsoa hore e be mocheso oa kamore pele e sebelisoa.

9.2.4Nomoro:Nomoro ea maemo a microwell 'me litekanyetso tsohle le lisampole li lokela ho tsamaisoa ka makhetlo a mabeli.Ngola maemo le lisampole maemo.

9.2.5Ho hlapolloa ha motsoako oa li-anti-anti-concentrated: hlapolla tharollo ea enzyme e tsitsitseng ka enzyme conjugate diluents ka tekanyo ea molumo oa 1:10 (1 fold concentrated enzyme solution: 10 folds enzyme conjugate diluents).

9.2.6Kenya tharollo e tloaelehileng / sampole le tharollo ea enzyme conjugate: eketsa 50µl ea tharollo e tloaelehileng kapa sampole e lokiselitsoeng lilibeng tse tsamaellanang, eketsa tharollo ea 50µl ea enzyme conjugate.Kopanya ka bonolo ka ho sisinya poleiti ka letsoho 'me u e kenye metsotso e 30 ho 25 ℃ ka sekoahelo.

9.2.7Hlatsoa: Tlosa sekoahelo ka bonolo 'me u tšele mokelikeli ka ntle ho liliba' me u hlatsoe li-microwells ka tharollo e hlatsoitsoeng ea 250µl (tharollo6) ka karohano ea 10s bakeng sa 4-5 linako tse ling.Monya metsi a setseng ka pampiri e monyang (bubble e setseng ea moea e ka felisoa ka ntlha e sa sebelisoeng).

9.2.8Mebala: Kenya 50µl substrate solution A le 50ul substrate solution B selibeng ka seng.Kopanya ka bonolo ka ho sisinya poleiti ka letsoho 'me u e kenye metsotso e 15 ho 25 ℃ ka sekoahelo (sheba 12.8).

9.2.11Lekanya: Kenya 50µl tharollo ea ho emisa selibeng ka seng.Kopanya ka bonolo ka ho sisinya poleiti ka letsoho 'me u lekanye monyetla ka 450nm (E khothalelitse tekanyo e nang le maqhubu a mabeli a 450/630nm. Bala sephetho nakong ea metsotso e 5 ka mor'a ho eketsa tharollo ea ho emisa.)

10 Liphetho

10.1Peresente ea ho monya

Litekanyetso tse tloaelehileng tsa litekanyetso tsa ho monya tse fumanoeng bakeng sa litekanyetso le lisampole li arotsoe ke boleng ba ho monya ba tekanyo ea pele (tekanyetso ea zero) 'me e atolosoa ka 100%.Tekanyetso ea zero ka hona e etsoa hore e lekane le 100% mme litekanyetso tsa ho monya li qotsitsoe ka liphesente.

=

B - - absorbance standard (kapa sampole)

B0 -- absorbance zero standard

10.2Mokokotlo o Tloaelehileng

----Ho etsa mokokotlo o tlwaelehileng: Nka boleng ba ho monya ba maemo joalo ka y-axis, semi logarithmic ea concentration ea AOZ standard solution (ppb) joalo ka x-axis.

----Maikutlo a AOZ a sampole e 'ngoe le e 'ngoe (ppb), e ka baloang ho tsoa lekotlong la calibration, e atisa ka ntlha e lumellanang ea dilution ea sampole e 'ngoe le e 'ngoe e lateloang, 'me bongata ba sampole boa fumanoa.

Ka kopo hlokomela:

Software e khethehileng e entsoe bakeng sa phokotso eohle ea data, e ka fanoang ka kopo.

Dilution factor………………………………………………………2

10.Kutloelo-bohloko, ho nepahala le ho nepahala

Kutloelo-bohloko: 0.025ppb

Moeli oa ho fumanoa:

Tishu (mesifa, sebete)……………………………….0.1ppb

Mahe a linotsi------------------------------------------------ -0.1ppb

Ho nepahala:

Lithishu tsa liphoofolo (mesifa le sebete)……………………100±20%

Mahe a linotsi…………………………………………………………………………. 100±20%

Nepahalo:CV ea ELISA kit e ka tlase ho 10%.

11.Hlokomela

12.1 Litekanyetso tse bolelang tsa litekanyetso tsa ho monya tse fumanoang bakeng sa litekanyetso le li-sampuli li tla fokotseha haeba li-reagents le li-sampuli li sa laoloe ho mocheso oa kamore (20-25℃).

12.2 Se ke oa lumella li-microwells ho omella lipakeng tsa mehato ho qoba ho se atlehe ho ikatisa le ho sebetsa mohato o latelang hang ka mor'a ho tlanya sets'oants'o sa li-microwell.

12.3 Tšoara reagent ka 'ngoe ka bonolo pele u e sebelisa.

12.4 Beha letlalo la hau hole le tharollo ea ho emisa hobane ke 0.5MH₂SO₄tharollo.

12.5 Se ke oa sebelisa lisebelisoa tse siiloeng ke nako.Se ke oa fapanyetsana li-reagents tsa lihlopha tse fapaneng, ho seng joalo e tla theola maikutlo.

12.6 Boemo ba polokelo: Boloka lisebelisoa tsa ELISA ho 2-8 ℃, u se ke oa hoama.Koahela lipoleiti tsa microwell tse phomolang, Qoba khanya ea letsatsi e otlolohileng nakong ea li-incubations tsohle.Ho khothalletsoa ho koahela lipoleiti tsa microtiter.

12.7 Tharollo ea substrate e lokela ho tloheloa haeba e fetola mebala.Li-reagents li ka 'na tsa senyeha haeba boleng ba ho monya (450/630nm) ba tekanyo ea zero bo le ka tlase ho 0.5 (A450nm<0.5).

12.8 Karabelo ea mebala e hloka 15min ka mor'a ho eketsoa ha tharollo ea substrate;U ka lelefatsa nako ea ho incubation ho 20min kapa ho feta haeba 'mala o le bobebe haholo hore o ka khethoa., le ka mohla u se ke ua feta 25min. Ho fapana le hoo, khutsufatsa nako ea ho qhoqhoa hantle.

12.9 Mocheso o nepahetseng oa ho arabela ke 25℃.Mocheso o phahameng kapa o tlase o tla lebisa liphetohong tsa kutloisiso le litekanyetso tsa ho monya.

12.Boemo ba polokelo le nako ea polokelo

Boemo ba polokelo: 2-8 ℃.

Nako ea polokelo: 12months.