huahana

1. 1.Kāpae

ʻO nā Nitrofurans nā lāʻau lapaʻau ākea ākea, i hoʻohana pinepine ʻia i ka hana holoholona no kāna mau antibacterial maikaʻi a me nā waiwai pharmacokinetic.Ua hoʻohana pū ʻia lākou ma ke ʻano he mea hoʻoulu ulu i ka puaʻa, ka moa a me ka hana wai.Ma nā haʻawina lōʻihi me nā holoholona lab i hōʻike ʻia ua hōʻike nā lāʻau lapaʻau makua a me kā lākou metabolites i nā ʻano carcinogenic a me mutagenic.Ua alakaʻi kēia i ka pāpā ʻana i nā nitrofurans no ka mālama ʻana i nā holoholona i hoʻohana ʻia no ka hana ʻana i ka meaʻai.Ua pāpā ʻia nā lāʻau nitrofuran furaltadone, nitrofurantoin a me nitrofurazone mai ka hoʻohana ʻana i ka hana holoholona meaʻai ma EU ma 1993, a ua pāpā ʻia ka hoʻohana ʻana o furazolidone i ka makahiki 1995.

Pono ka nānā ʻana i ke koena o nā lāʻau nitrofuran ma muli o ka ʻike ʻana i nā metabolites paʻa i ka ʻiʻo o nā lāʻau makua nitrofuran.Ma muli o ka wikiwiki o ka hoʻololi ʻana o nā lāʻau lapaʻau makua, a e paʻa ana nā metabolites nitrofuran i hoʻopaʻa ʻia no ka manawa lōʻihi, ua hoʻohana ʻia kēia mau metabolites i mea e ʻike ai i ka hoʻomāinoino ʻana i nā nitrofurans, ʻo ia hoʻi me Furazolidone metabolite (AOZ), Furaltadone metabolite (AMOZ). ), Nitrofurantoin metabolite (AHD) a me Nitrofurazone metabolite (SEM).

Hoʻoholo pinepine ʻia nā koena AOZ e LC-MS a i ʻole LC-MS/MS.ʻO nā enzyme immunoassays, i hoʻohālikelike ʻia me nā ʻano chromatographic, hōʻike i nā pōmaikaʻi nui e pili ana i ka naʻau, ka palena ʻike, nā mea ʻenehana a me nā koi manawa.(Kina manawa: 45min)

1. 2.Kumu Hoao

Hoʻolālā ʻia kēia pahu ELISA e ʻike iā AOZ ma muli o ke kumu o ka immunoassay enzyme indirect-competitive.Ua uhi ʻia nā pūnāwai microtiter me ka antigen pili i ka BSA.Hoʻokūkū ʻo AOZ i ka laʻana me ka antigen i uhi ʻia ma ka pā microtiter no ka mea i hoʻohui ʻia.Ma hope o ka hoʻohui ʻana o ka enzyme conjugate, hoʻohana ʻia ka substrate chromogenic a ana ʻia ka hōʻailona e kahi spectrophotometer.Hoʻohālikelike ʻia ka absorption i ka ʻike AOZ i ka hāpana.

1. 3.Nā noi

Hiki ke hoʻohana ʻia kēia pahu i ka helu helu a me ka qualitative o ke koena AOZ i lokokino kino(ʻiʻo, ate etc.), meli.

1. 4.Nā hoʻololi keʻa

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.Pono Pono

5.1Mea lako

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

---- Rotary evaporator a i ʻole nā ​​mea hana hoʻomaloʻo nitrogen

----Homogenizer / ʻōpū

----Hololu

---- Mea hui pu me ka vortex

----Centrifuge

----Ke kaulike analytical (inductance: 0.01g)

----Pikipika puka puka: 10ml

----Upu paipu Rubber

----Paniani nui: 100ml

----Paniani aniani: 10ml

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

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

250ul-multipipette

5.2Nā mea hoʻopaʻa

----Ethyl acetate (AR)

----n-hexane (a i ʻole n-heptane) (AR)

----Dipotassium hydrogen phosphate trihydrate

(K₂HPO₄.3H₂ʻO) (AR)

----Akika hydrochloric paʻa (HCl, AR)

-----Methanol

----Sodium hydroxide (NaOH, AR)

----Wai deionized

1. 6.Nā ʻāpana pahu

l ka pā Microtiter i uhi ʻia me ka antigen, 96 luawai

l Nā hāʻina maʻamau (6 ʻōmole, 1ml / ʻōmole)

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

l Spiking mana maʻamau: (1ml/'ōmole)........100ppb

l Hoʻohui ʻia ka enzyme conjugate 1ml……..pale ʻulaʻula

l Nā mea hoʻoheheʻe conjugate Enzyme 10ml………..palena ʻōmaʻomaʻo

l Lapaʻa A 7ml………………………………………………..pale keʻokeʻo

l Pāpala B 7ml………………………………………………..pale ʻulaʻula

l Hooki i ka solution 7ml……………………………………

l 20× hoʻonā holoi holoi 40ml

……………………………………………..pale mālohi

l 2 × hoʻonā hoʻoheheʻe ʻia 60ml….palena uliuli

l 2-Nitrobenzaldehyde 15.1mg………………keokeo

1. 7.Hoʻomākaukau Reagents

Hoʻoholo 1: mea hoʻoheheʻe derivative:

E hoʻohui i 10ml o ka methanol i ka ʻōmole me 2-Nitrobenzaldehyde a hoʻoheheʻe.(ma ke kumu o 10mM).

Hoʻoholo 2: 0.1MK₂HPO₄hoʻonā:

Kaumaha 22.8g K₂HPO₄.3H₂O i 1L o ka wai deionized e hoʻoheheʻe.

Hoʻoholo 3: 1M HCl solution

E hoʻoheheʻe i ka waika hydrochloric concentrated 8.3ml me ka wai deionized i 100ml.

Pane 4:1M NaOH solution

E hoʻonā i ka 4g NaOH me 100ml wai deionized.

Haʻina 5: hoʻonā ʻili:

E hoʻoheheʻe i ka wai hoʻoheheʻe 2×concentrated me ka wai deionized i ka lakene o 1:1.Hiki ke mālama ʻia kēia hoʻonā no 1 mahina ma 4 ℃, e hoʻohana ʻia no ka unuhi ʻana i nā laʻana.

Hoʻoholo 6: holoi holoi:

E hoʻoheheʻe i ka wai holoi holoi 20xconcentrated me ka wai deionized i ka nui o ka ration o 1:19, e hoʻohana ʻia e holoi i nā papa.Hiki ke mālama ʻia kēia hopena i hoʻoheheʻe ʻia no 1 mahina ma 4 ℃.

1. 8.Hoʻomākaukau Laʻana

8.1Hoʻomaopopo a mālama mua i ka hana:

a) E ʻoluʻolu e hoʻohana i nā ʻōlelo aʻoaʻo hoʻokahi i ka hoʻokolohua, a hoʻololi i nā ʻōlelo aʻoaʻo i ka wā e komo ai i nā reagent ʻē aʻe.

b) E hōʻoia i ka maʻemaʻe o nā mea hana hoʻokolohua.

c) hiki ke mālama ʻia ka reagent derivative ma 2-8 ℃ no ʻekolu mahina;

d) Hiki ke mālama 'ia ka solution HCl ma ka lumi wela no 3 mahina;

e) Hiki ke mālama ʻia ka hoʻonā NaOH no 3 mahina ma ke ana wela;

f) E mālama i nā laʻana i mālama ʻole ʻia i loko o ka hau (-20 ℃);

g) Hiki ke mālama ʻia nā laʻana i mālama ʻia no 24h ma 2-8 ℃ i ka pōʻeleʻele.

8.2 Nā laʻana o nā ʻiʻo holoholona a me nā ate:

---- Homogenize i nā laʻana me ka homogenizer;

---- Kaumaha 1.0±0.05g o ka homogenized kiko hāpana i loko o 50ml polystyrene centrifuge paipu.E hoʻohui i 4ml wai deionized, 0.5ml 1M HCl solution a me 100ul derivative reagent (e nānā i ka hopena 1).E lulu ia no 2min.

---- Incubate ma 37 ℃ i ka pō (ma kahi o 16h);

---- Hoʻohui 5ml 0.1MK₂HPO₄ (hoʻonā2), 0.4ml 1M NaOH (hoʻonā4) a me 5ml ethyl acetate.E lulu ikaika no 30s;

---- Centrifuge ma ka lumi wela (20-25 ℃) no 10min, ma kahi o 3000g;

---- E lawe i ka 2.5ml o ka supernatant organic phase i loko o ka 10ml maʻemaʻe ipu aniani, maloʻo me 50-60℃ kinoea hau a i ʻole rotary evaporator;

---- E hoʻoheheʻe i ke koena maloʻo me 1ml n-hexane (a i ʻole n-heptane), vortex no 30s, e hoʻohui i 1ml solution extraction (hoʻonā5), vortex 1min, hui piha.

---- Centrifuge ma ka lumi wela (20-25^oC) no 5min, ma kahi o 3000g;

---- Wehe i ka ʻāpana organik supernatant;e lawe i 50μl o ka pae wai substrate no ka hoʻāʻo.

8.4 Meli

----kaumaha 1.0 ± 0.05g o ka homogenized honey hāpana i loko o ka 50ml polystyrene centrifuge paipu;

---- Hoʻohui i 4ml wai deionized, 0.5ml 1M HCl (hoʻonā3) a me 100μl derivative reagent (hoʻonā1);e lulu loa no 2min;

----Incubate ma 37 ℃ i ka pō (ma kahi o 16h);

----Hoʻohui 5ml 0.1MK₂HPO₄ (hoʻonā2), 0.4ml 1M NaOH (hoʻonā4) a me 5ml ethyl acetate, lulu ikaika no 30s;

----Centrifuge ma ka lumi wela (20-25 ℃) no 10min, ma ka liʻiliʻi loa 3000g;

----E lawe i ka 2.5ml o ka supernatant organic phase i loko o ka 10ml maʻemaʻe ipu aniani, maloʻo me 50-60℃ kinoea hau a i ʻole rotary evaporator;

---- E hoʻoheheʻe i ka mea maloʻo me ka 1ml n-hexane (a i ʻole n-heptane), vortex no 30s, e hoʻohui i 1ml solution extraction (hoʻonā5), vortex 1min, hui piha.

----Centrifuge ma ka lumi wela (20-25^oC) no 10min, ma kahi o 3000g;

---- Wehe i ka mea hoʻolāʻau supernatant;e lawe i 50μl o ka pae wai substrate no ka hoʻāʻo.

1. 9.Kaʻina hoʻokolohua

9.1Hoʻomaopopo ma mua o ka hoʻāʻo ʻana

9.1.1 E hoʻopaʻa pono i nā reagents a me nā microwell a pau ma ka lumi wela (20-25 ℃).

9.1.2 E hoʻihoʻi i nā mea hou i koe i 2-8 ℃ ma hope koke o ka hoʻohana ʻana.

9.1.3 ʻO ka holoi pono ʻana i nā microwells kahi hana koʻikoʻi i ke kaʻina hana hoʻāʻo;ʻo ia ka mea koʻikoʻi i ka reproducibility o ka ELISA analysis.

9.1.4 E pale i ka malamalama a e uhi i na microwells i ka wa e hoohua ai.

9.2 Nā ʻanuʻu hōʻike

9.2.1 E lawe i nā mea hou a pau i waho ma ka lumi wela (20-25 ℃) no ka ʻoi aku ma mua o 30min, homogenize ma mua o ka hoʻohana ʻana.

9.2.2 E kiʻi i nā microwells pono a hoʻihoʻi i ke koena i loko o ka ʻeke paʻa zip ma 2-8 ℃ koke.

9.2.3 Pono e hoʻomaʻamaʻa hou ʻia ka wai holoi holoi concentrated a me ka solution extraction concentrated i ka mahana wela ma mua o ka hoʻohana ʻana.

9.2.4Helu:E helu ʻia kēlā me kēia kūlana microwell a me nā kūlana a me nā laʻana a pau e holo pālua.E hoʻopaʻa i nā kūlana maʻamau a me nā laʻana.

9.2.5ʻO ka hoʻoheheʻe ʻana o ka hoʻonā antibody concentrated: hoʻoheheʻe i ka hopena enzyme concentrated me nā mea hoʻoheheʻe conjugate enzyme ma ka ratio o ka leo o 1:10(1 pānaʻi hoʻonā enzyme concentrated: 10 mau mea hoʻoheheʻe conjugate enzyme).

9.2.6Hoʻohui i ka hoʻonā maʻamau / hāpana a me ka hopena conjugate enzyme: hoʻohui i 50µl o ka hopena maʻamau a i ʻole ka laʻana i hoʻomākaukau ʻia i nā luawai pili, e hoʻohui i ka solution conjugate enzyme 50µl.E hoʻokuʻi mālie me ka lulu ʻana i ka pā me ka lima a hoʻomoʻa no 30 mau minuke ma 25 ℃ me ka uhi.

9.2.7Holoi: Wehe mālie i ka uhi a ninini i ka wai mai loko mai o nā pūnāwai a holoi i nā microwells me 250µl wai holoi holoi (hoʻonā6) ma kahi o 10s no 4-5 mau manawa.E hoʻopau i ke koena wai me ka pepa hoʻoheheʻe (hiki ke hoʻopau ʻia ke koena o ka ea me ka ʻaoʻao i hoʻohana ʻole ʻia).

9.2.8kala kala: E hoʻohui i 50µl substrate solution A me 50ul substrate solution B i kēlā me kēia pūnāwai.Hoʻohui mālie me ka hoʻoluliluli ʻana i ka pā me ka lima a hoʻomoʻa no 15min ma 25 ℃ me ka uhi (e nānā i ka 12.8).

9.2.11Ana: E hoʻohui i 50µl i ka hopena hoʻomaha i kēlā me kēia pūnāwai.Hoʻohui mālie me ka ʻoluʻolu ʻana i ka pā me ka lima a ana i ka absorbance ma 450nm (Ua manaʻo ʻia e ana me ka lōʻihi ʻelua o 450/630nm. Heluhelu i ka hopena i loko o 5min ma hope o ka hoʻohui ʻana i ka solution stop.)

10 Nā hualoaʻa

10.1Pākēneka o ke komo ʻana

Hoʻokaʻawale ʻia nā kumukūʻai kumu o nā waiwai absorbance i loaʻa no nā maʻamau a me nā hōʻailona e ka waiwai absorbance o ka maʻamau mua (zero standard) a hoʻonui ʻia e 100%.No laila, ua like ka zero standard me 100% a ua helu ʻia nā helu absorbance ma nā pākēneka.

=

B ——absorbance maʻamau (a i ʻole ka laʻana)

B0 ——absorbance zero standard

10.2Pipi maʻamau

----E kahakii i ka pihi maʻamau: E lawe i ka waiwai hoʻopaneʻe o nā maʻamau ma ke ʻano he axis-y, semi logarithmic o ka neʻe ʻana o ka hopena maʻamau AOZ (ppb) ma ke ʻano he-x.

---- Ua hoonuiia ka AOZ o kela a me keia lapana (ppb), i hiki ke heluheluia mai ke kaha o ka hoohalike ana, e ka mea hookahe hoohalike like o kela me keia lapana i ukaliia, a ua loaa mai ka manao maoli o ka laana.

E ʻoluʻolu e hoʻomaopopo:

Ua hoʻomohala ʻia nā polokalamu kūikawā no ka hōʻemi ʻikepili āpau, hiki ke hāʻawi ʻia ma ke noi.

Mea hoʻoheheʻe………………………………………………………2

10.ʻIke, pololei a me ka pololei

ʻO ka ʻike: 0.025ppb

palena ʻike:

Tissue (ʻiʻo, ate)………………………………0.1ppb

Mele------------------------------------------------- -0.1ppb

pololei:

Kaʻiʻo holoholona (ka ʻiʻo a me ke akepaʻa)……………………100±20%

Mele…………………………………………………… 100±20%

pololei:ʻO ka CV o ka pahu ELISA he emi iho ma mua o 10%.

11.Hoolaha

12.1 E hoʻemi ʻia nā kumu waiwai o nā waiwai absorbance i loaʻa no nā maʻamau a me nā laʻana inā ʻaʻole i hoʻoponopono ʻia nā reagents a me nā laʻana i ka lumi wela (20-25 ℃).

12.2 Mai ʻae i nā microwells e maloʻo ma waena o nā ʻanuʻu e pale i ka hana hou ʻole ʻana a hana i ka ʻanuʻu aʻe ma hope koke o ka paʻi ʻana i ka mea paʻa microwells.

12.3 E lulu mālie i kēlā me kēia reagent ma mua o ka hoʻohana ʻana.

12.4 E hoʻokaʻawale i kou ʻili mai ka hopena hoʻomaha no ka mea ʻo ia ka 0.5MH₂SO₄hoʻonā.

12.5 Mai hoʻohana i nā kit i ka wā kahiko.Mai hoʻololi i nā reagents o nā pūʻulu like ʻole, a i ʻole e hāʻule ka naʻau.

12.6 Kūlana mālama: E mālama i nā pahu ELISA ma 2-8 ℃, mai maloʻo.Hoʻopaʻa i nā papa microwell hoʻomaha, E pale pololei i ka lā i ka wā e hoʻoulu ai.Paipai ʻia ka uhi ʻana i nā papa microtiter.

12.7 E haʻalele ʻia ka hoʻonā substrate inā e ʻano kala.Hiki ke hōʻemi ʻia nā reagents inā ʻoi aku ka liʻiliʻi o ka waiwai absorbance (450/630nm) o ka zero ma mua o 0.5 (A450nm<0.5).

12.8 Pono ka hopena kala i 15min ma hope o ka hoʻohui ʻana o ka hopena substrate;Hiki iā ʻoe ke hoʻolōʻihi i ka manawa incubation i 20min a ʻoi aʻe paha inā ʻoi aku ka māmā o ke kala e hoʻoholo ʻia., Mai ʻoi aku ma mua o 25min. Ma ka mea ʻē aʻe, e hoʻopōkole pono i ka manawa incubation.

12.9 ʻO ka mahana hopena maikaʻi loa he 25 ℃.ʻO ke kiʻekiʻe a i ʻole ka haʻahaʻa haʻahaʻa e alakaʻi i nā loli o ka naʻau a me nā waiwai absorbance.

12.Ke kūlana mālama a me ka manawa mālama

Kūlana mālama: 2-8 ℃.

Ka manawa mālama: 12 mau mahina.