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Ngwa Enzyme Immunoassay nke asọmpi maka nyocha ọnụọgụ nke Furazolidone metabolite (AOZ)

Nkọwa dị mkpirikpi:

Emebere ngwa ELISA a iji chọpụta AOZ dabere na ụkpụrụ nke immunoassay enzyme na-abụghị nke asọmpi.A na-ekpuchi olulu mmiri microtiter na antigen jikọtara BSA.AOZ na sample na-asọmpi na antigen mkpuchi na efere microtiter maka mgbochi agbakwunyere.Mgbe mgbakwunye nke enzyme conjugate, a na-eji mkpụrụ chromogenic eme ihe ma jiri spectrophotometer tụọ mgbaàmà.Ntinye ahụ na-agbanwe agbanwe na ntinye uche AOZ na nlele.


Nkọwa ngwaahịa

Mkpado ngwaahịa

Ngwa Enzyme Immunoassay nke asọmpi maka

Quantitative Analysis nkeFurazolidone metabolite(AOZ)

 

  1. 1.ndabere

Nitrofurans bụ ọgwụ nje ndị na-emepụta ihe na-eme ka a na-emepụta ihe, bụ nke a na-ejikarị eme ihe na mmepụta anụmanụ maka ezigbo antibacterial na pharmacokinetic Njirimara.A na-ejikwa ha dị ka ndị na-akwalite uto na ezì, anụ ọkụkọ na mmepụta mmiri.N'ime ogologo oge ọmụmụ na anụmanụ ụlọ nyocha gosiri na ọgwụ ndị nne na nna na metabolites ha gosipụtara àgwà carcinogenic na mutagenic.Nke a emeela ka amachibidoro nitrofurans maka ọgwụgwọ anụmanụ eji eme ihe oriri.A machibidoro ọgwụ nitrofuran furaltadone, nitrofurantoin na nitrofurazone iji na-emepụta anụmanụ nri na EU na 1993, machibido iji furazolidone na 1995.

Nyocha nke ihe fọdụrụ na ọgwụ nitrofuran kwesịrị ịdabere na nchọpụta nke anụ ahụ na-ejikọta metabolites nke ọgwụ nne na nna nitrofuran.Ebe ọ bụ na ọgwụ ndị nne na nna na-emewanye ngwa ngwa, na nitrofuran metabolites na-ejikọta anụ ahụ ga-ejigide ogologo oge, a na-eji metabolites ndị a dị ka ihe mgbaru ọsọ na nchọpụta nke mmetọ nke nitrofuran, nke gụnyere Furazolidone metabolite (AOZ), Furaltadone metabolite (AMOZ). Nitrofurantoin metabolite (AHD) na nitrofurazone metabolite (SEM).

AOZ- residues na-ekpebikarị site na LC-MS ma ọ bụ LC-MS/MS.Enzyme immunoassays, ma e jiri ya tụnyere ụzọ chromatographic, na-egosi uru dị ukwuu gbasara uche, oke nchọpụta, akụrụngwa teknụzụ na oge achọrọ.(Oge Ọnụ: 45min)

  1. 2.Ụkpụrụ ule

Emebere ngwa ELISA a iji chọpụta AOZ dabere na ụkpụrụ nke immunoassay enzyme na-abụghị nke asọmpi.A na-ekpuchi olulu mmiri microtiter na antigen jikọtara BSA.AOZ na sample na-asọmpi na antigen mkpuchi na efere microtiter maka mgbochi agbakwunyere.Mgbe mgbakwunye nke enzyme conjugate, a na-eji mkpụrụ chromogenic eme ihe ma jiri spectrophotometer tụọ mgbaàmà.Ntinye ahụ na-agbanwe agbanwe na ntinye uche AOZ na nlele.

  1. 3.Ngwa

Enwere ike iji ngwa a na nyocha ọnụọgụ na qualitative nke AOZ residue inanụ ahụ anụ ahụ(anụ ahụ, imeju wdg), mmanụ aṅụ.

  1. 4.Mmeghachi omume

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

Furaltadone metabolite (AMOZ) …………………………………………………………………….

Nitrofurantoin metabolite (AHD) …………………………………

Nitrofurazone metabolite (SEM) …………………………………………………………………….

Furazolidone …………………………………………………………………………………………………………

Furaltadone …………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………….

Nitrofurantoin……………………………………………………………………….

Nitrofurazone………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………….

  1. 5.Ihe achọrọ

5.1Akụrụngwa

---- Mpekere spectrophotometer nke Microtiter (450nm/630nm)

---- Rotary evaporator ma ọ bụ akụrụngwa ihicha nitrogen

----Homogenizer / afọ

----Shaker

---- igwekota Vortex

----Centrifuge

---- Ntụle nyocha (inductance: 0.01g)

---- pipette emechara: 10ml

---- bọlbụ pipette

---- Mpekere olu: 100ml

---- Mpekere iko: 10ml

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

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

250ul-multipipette

5.2Reagents

----Ethyl acetate (AR)

----n-hexane (ma ọ bụ n-heptane) (AR)

----Dipotassium hydrogen phosphate trihydrate

(K2HPO4.3H2O) (AR)

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

--Methanol

----Sodium hydroxide (NaOH, AR)

----Mmiri a na-agbapụta

  1. 6.Ngwa ngwa

l Microtiter efere kpuchie na antigen, olulu mmiri 96

l Standard ngwọta (6 karama, 1ml / karama)

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

l Spiking ọkọlọtọ njikwa: (1ml / karama).......100pb

l concentrated enzyme conjugate 1ml ...... okpu uhie

l Enzyme conjugate diluents 10ml……….. okpu akwụkwọ ndụ akwụkwọ ndụ

l Substrate A 7ml………………………………………………………………. okpu ọcha

l Substrate B 7ml………………………………………………………. okpu uhie

l Kwụsị ngwọta 7ml………………………………………………………………………. okpu edo edo

l 20 × lekwasị asacha ngwọta 40ml

…………………………………………………………………………………………………………………………

l 2 × ndọpụ mmịpụta ngwọta 60ml….-acha anụnụ anụnụ okpu

l 2-Nitrobenzaldehyde 15.1mg………………………. okpu ọcha

  1. 7.Nkwadebe Reagent

Ngwọta 1: ihe nrụpụta reagent:

Tinye 10ml nke methanol na karama nwere 2-Nitrobenzaldehyde na igbari.(na ntinye nke 10mM).

Ngwọta 2: 0.1MK2HPO4ngwọta:

Arọ 22.8g K2HPO4.3H2O ruo 1L nke mmiri deionized ka ọ gbazee.

Ngwọta 3: 1M HCl ngwọta

Igbari 8.3ml concentrated hydrochloric acid na deionized mmiri na 100ml.

Ngwọta 4:1M NaOH ngwọta

Gbarie 4g NaOH na mmiri 100ml deionized.

Ngwọta 5: ngwọta mmịpụta:

Dilute 2 × lekwasị mmịpụta ngwọta na deionized mmiri na olu ruru nke 1:1.Nke a ngwọta nwere ike chekwa maka 1month na 4 ℃, nke a ga-eji amịpụtara samples.

Ngwọta 6: ngwọta saa:

Ghichaa ihe ngwọta nke 20 × na-ekpo ọkụ na mmiri na-ekpo ọkụ na oke olu nke 1:19, nke a ga-eji na-asa efere.Nke a diluted ngwọta nwere ike chekwa maka 1 ọnwa na 4 ℃.

  1. 8.Nkwadebe ihe atụ

8.1Ọkwa na akpachapụ anya tupu arụ ọrụ:

a) Biko jiri ndụmọdụ na-apụ apụ na nnwale ahụ, ma gbanwee ndụmọdụ mgbe ị na-etinye reagent dị iche iche.

b) Gbaa mbọ hụ na ngwa nnwale niile dị ọcha.

c) enwere ike ichekwa reagent ihe na 2-8 ℃ maka ọnwa atọ;

d) Enwere ike ịchekwa ngwọta HCl na ụlọ okpomọkụ maka ọnwa 3;

e) Enwere ike ịchekwa ngwọta NaOH maka ọnwa 3 na okpomọkụ ụlọ;

f) Debe ihe nlele a na-emebeghị na friza (-20 ℃);

g) Enwere ike ichekwa ihe nlele emere maka 24h na 2-8 ℃ n'ọchịchịrị.

8.2 Ihe nlele anụ ahụ na imeju:

----Homogenize ndị samples na homogenizer;

---- Ibu 1.0 ± 0.05g nke anụ ahụ homogenized n'ime 50ml polystyrene centrifuge tube.Tinye 4ml deionized mmiri, 0.5ml 1M HCl ngwọta na 100ul derivative reagent (lee ngwọta 1).Magharịa ya maka nkeji 2.

---- Incubate na 37 ℃ n'abali (ihe dị ka elekere 16);

---- Tinye 5ml 0.1MK2HPO4 (ngwọta2), 0.4ml 1M NaOH (ngwọta4) na 5ml ethyl acetate.Ma jijiji nke ukwuu maka afọ 30;

---- Centrifuge na ụlọ okpomọkụ (20-25 ℃) maka 10min, ọ dịkarịa ala 3000g;

---- Were 2.5ml nke supernatant organic oge n'ime a 10ml ọcha iko tube, kpọrọ nkụ na 50-60 ℃ nitrogen gas ma ọ bụ rotary evaporator;

---- Gbarie akọrọ akọrọ na 1ml n-hexane (ma ọ bụ n-heptane), vortex maka 30s, gbakwunye ngwọta mmịpụta 1ml (ngwọta5), vortex 1min, mix kpamkpam.

---- Centrifuge na ụlọ okpomọkụ (20-25oC) maka 5min, ọ dịkarịa ala 3000g;

---- Wepu akụkụ nke organic na-enweghị atụ;Were 50μl nke mkpụrụ osisi mmiri maka nyocha.

 

8.4 Mmanụ aṅụ

----atụ 1.0 ± 0.05g nke ihe nlele mmanụ aṅụ homogenized n'ime tube centrifuge 50ml polystyrene;

---- Tinye 4ml mmiri deionized, 0.5ml 1M HCl (ngwọta3) na 100μl ewepụtara reagent (ngwọta1);maa jijiji kpamkpam maka nkeji 2;

----Incubate na 37 ℃ n'abali (ihe dị ka awa 16);

---- Tinye 5ml 0.1MK2HPO4 (ngwọta2), 0.4ml 1M NaOH (ngwọta4) na 5ml ethyl acetate, maa jijiji nke ukwuu maka 30s;

----Centrifuge na ụlọ okpomọkụ (20-25 ℃) maka 10min, ọ dịkarịa ala 3000g;

---- Were 2.5ml nke supernatant organic oge n'ime a 10ml ọcha iko tube, kpọrọ nkụ na 50-60 ℃ nitrogen gas ma ọ bụ rotary evaporator;

---- Wepu ihe akọrọ akọrọ na 1ml n-hexane (ma ọ bụ n-heptane), vortex maka 30s, tinye ngwọta mmịpụta 1ml (ngwọta5), vortex 1min, mix kpamkpam.

----Centrifuge na ụlọ okpomọkụ (20-25oC) maka 10min, ọ dịkarịa ala 3000g;

----Wepụ akụkụ ahụ nke dị elu;Were 50μl nke mkpụrụ osisi mmiri maka nyocha.

  1. 9.Usoro nyocha

9.1Rịba ama tupu nyocha

9.1.1 Jide n'aka na ihe niile reagents na microwells niile dị na ụlọ okpomọkụ (20-25 ℃).

9.1.2 Weghachite ihe niile fọdụrụ reagents ka 2-8 ℃ ozugbo eji.

9.1.3 Ịsacha microwells nke ọma bụ nzọụkwụ dị mkpa na usoro nyocha;Ọ bụ ihe dị mkpa maka mmụgharị nke nyocha ELISA.

9.1.4 Zere ọkụ ma kpuchie microwells n'oge incubation.

9.2 Usoro nyocha

9.2.1 Wepụ ihe niile reagents na ụlọ okpomọkụ (20-25 ℃) maka ihe karịrị 30min, homogenize tupu ojiji.

9.2.2 Nweta microwells achọrọ wee weghachi ndị ọzọ n'ime akpa mkpọchi zip na 2-8 ℃ ozugbo.

9.2.3 A ga-emegharị ihe ngwọta na-asachapụ nke na-etinye uche na ngwọta mmịpụta ka ọ nọrọ n'ime ụlọ okpomọkụ tupu ejiri ya.

9.2.4Nọmba:Agụnyere ọnọdụ microwell ọ bụla yana ụkpụrụ na nlele niile kwesịrị ịme ya na oyiri.Dekọọ ụkpụrụ na ọnọdụ nlele.

9.2.5Dilution nke ngwọta mgbochi mmadụ gbadoro anya: itughari ihe ngwọta nke enzyme na-etinye uche na enzyme conjugate diluents na oke olu nke 1:10 (1 fold concentrated enzyme solution: 10 folds enzyme conjugate diluents).

9.2.6Tinye ọkọlọtọ ọkọlọtọ / sample na ngwọta conjugate enzyme: tinye 50µl nke ọkọlọtọ ọkọlọtọ ma ọ bụ ihe nlele akwadoro na olulu mmiri kwekọrọ, tinye 50µl ngwọta conjugate enzyme.Gwakọta nke ọma site na iji aka na-amagharị efere ahụ ma tinye ya maka 30min na 25 ℃ na mkpuchi.

9.2.7saa: Wepu mkpuchi ahụ nke ọma ma wụsa mmiri mmiri na olulu mmiri ma kpochaa microwells na 250µl diluted wash solution (ngwọta6) na etiti oge nke 10s maka oge 4-5.Were akwụkwọ na-amịkọrọ mmiri na-amịpụta mmiri fọdụrụnụ (enwere ike iwepụ afụ ikuku ndị ọzọ site n'ọnụ ọnụ na-ejighị ya).

9.2.8Agba: Tinye 50µl ngwọta A na 50ul substrate solution B na nke ọ bụla.Gwakọta nke ọma site na iji aka na-amagharị efere ahụ ma tinye ya maka 15min na 25 ℃ na mkpuchi (lee 12.8).

9.2.11tụọ: Tinye 50µl ngwọta nkwụsị na olulu mmiri ọ bụla.Gwakọta nke ọma site na iji aka na-amagharị efere ahụ wee tụọ nnabata ahụ na 450nm (Ọ tụrụ aro ka ọ bụrụ ogologo ihu abụọ nke 450/630nm. Gụọ nsonaazụ ya n'ime 5min mgbe mgbakwunye nke ngwọta nkwụsị.)

10 Nsonaazụ

10.1Ntinye pasentị

The pụtara ụkpụrụ nke absorbance ụkpụrụ enwetara maka ụkpụrụ na samples na-ekewa site absorbance uru nke mbụ ọkọlọtọ (efu ọkọlọtọ) na mụbaa site 100%.A na-eme ọkọlọtọ efu ka ọ bụrụ 100% yana ụkpụrụ nnabata na-ehota na pasentị.

=

B ——ụkpụrụ nnabata (ma ọ bụ sample)

B0 ——ụkpụrụ efu efu

10.2Usoro ọkọlọtọ

----Iji see a ọkọlọtọ usoro: Were absorbance uru nke ụkpụrụ dị ka y-axis, ọkara logarithmic nke ịta nke AOZ ọkọlọtọ ngwọta (ppb) dị ka x-axis.

---- AOZ ntinye uche nke ihe nlele ọ bụla (ppb), nke enwere ike ịgụ site na nhazi nhazi, na-abawanye site na ihe ntinye dilution kwekọrọ na nke ọ bụla na-esote, ma nweta ezigbo ntinye nke sample.

Biko mara:

Emepụtara sọftụwia pụrụ iche maka mbelata data niile, nke enwere ike ịnye na arịrịọ.

Ihe dilution…………………………………………………………………………………

10.Mmetụta, izi ezi na nkenke

Mmetụta: 0.025pb

Oke nchọpụta:

Anụ ahụ (anụ ahụ, imeju)………………………………………………………………..1ppb

Mmanụ aṅụ ---------------------------------- -0.1 pb

izi ezi:

anụ ahụ anụ ahụ (akwara na imeju) ……………………………………………………………… 100± 20%

Mmanụ aṅụ……………………………………………………………………………………………… 100±20%

Nkenkenke:CV nke ngwa ELISA erughị 10%.

11.Mara

12.1 The pụtara ụkpụrụ nke absorbance ụkpụrụ enwetara maka ụkpụrụ na samples ga-ebelata ma ọ bụrụ na reagents na samples e regulated ka ụlọ okpomọkụ (20-25 ℃).

12.2 Ekwela ka microwells kpoo n'etiti usoro iji zere mmeghari na-enweghị ihe ịga nke ọma ma rụọ ọrụ na-esote ozugbo ka ịpịa ihe njide microwells.

12.3 Kwanye reagent nke ọ bụla nwayọ tupu eji ya.

12.4 Debe akpụkpọ gị pụọ na ngwọta nkwụsị n'ihi na ọ bụ 0.5MH2SO4ngwọta.

12.5 Ejila ngwa ndị ahụ emechiela.Agbanwela reagents nke batches dị iche iche, ma ọ bụghị ya, ọ ga-agbadata uche.

12.6 Ọnọdụ nchekwa: Debe ngwa ELISA na 2-8 ℃, ajụla oyi.Mechie efere microwell izu ike, Zere ìhè anyanwụ kwụ ọtọ n'oge nnabata niile.A na-atụ aro ikpuchi efere microtiter.

12.7 kwesịrị ịhapụ ihe ngwọta nke mkpụrụ osisi ma ọ bụrụ na ọ gbanwee agba.The reagents nwere ike njọ ma ọ bụrụ na absorbance uru (450/630nm) nke efu ọkọlọtọ bụ ihe na-erughị 0.5 (A450nm <0.5).

12.8 Mmeghachi omume agba chọrọ 15min mgbe mgbakwunye nke ngwọta mkpụrụ;Ị nwere ike ịgbatị oge ntinye ahụ ruo 20min ma ọ bụ karịa ma ọ bụrụ na agba dị oke ọkụ nke a na-apụghị ikpebi., Agafeghị 25min. Kama nke ahụ, belata oge ntinye nke ọma.

12.9 ezigbo mmeghachi omume okpomọkụ bụ 25 ℃.Okpomọkụ dị elu ma ọ bụ dị ala ga-eduga na mgbanwe nke uche na ụkpụrụ nnabata.

12.Ọnọdụ nchekwa na oge nchekwa

Ọnọdụ nchekwa: 2-8 ℃.

Oge nchekwa: 12 ọnwa.

 

 


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