mankhwala

1. 1.Mbiri

Nitrofuran ndi mankhwala opha tizilombo tosiyanasiyana, omwe amagwiritsidwa ntchito kaŵirikaŵiri popanga zinyama chifukwa cha mankhwala ake abwino kwambiri a antibacterial ndi pharmacokinetic.Amagwiritsidwanso ntchito ngati kulimbikitsa kukula kwa nkhumba, nkhuku ndi kupanga zam'madzi.M'kupita kwa nthawi, maphunziro a labu nyama anasonyeza kuti makolo mankhwala ndi metabolites awo anasonyeza carcinogenic ndi mutagenic makhalidwe.Izi zapangitsa kuti ma nitrofuran aletsedwe pochiza nyama zomwe zimagwiritsidwa ntchito popanga chakudya.Mankhwala a nitrofuran a furaltadone, nitrofurantoin ndi nitrofurazone analetsedwa kugwiritsidwa ntchito popanga nyama ku EU mu 1993, ndipo kugwiritsa ntchito furazolidone kunali koletsedwa mu 1995.

Kuwunika kwa zotsalira za nitrofuran kuyenera kutengera kuzindikira kwa ma metabolites omangidwa ndi minofu ya mankhwala a nitrofuran.Popeza mankhwala a makolo amapangidwa mwachangu kwambiri, ndipo minofu yomangidwa ndi nitrofuran metabolites idzasungidwa kwa nthawi yayitali, ma metaboliteswa amagwiritsidwa ntchito ngati chandamale pozindikira kuzunzidwa kwa nitrofurans, kuphatikiza Furazolidone metabolite (AOZ), Furaltadone metabolite (AMOZ). ), Nitrofurantoin metabolite (AHD) ndi Nitrofurazone metabolite (SEM).

Zotsalira za AOZ zimatsimikiziridwa nthawi zambiri ndi LC-MS kapena LC-MS/MS.Ma enzyme immunoassays, poyerekeza ndi njira za chromatographic, amawonetsa zabwino zambiri zokhuza kukhudzika, malire ozindikira, zida zamakono komanso nthawi yofunikira.(Nthawi mtengo: 45min)

1. 2.Mfundo Yoyesera

Chida ichi cha ELISA chapangidwa kuti chizindikire AOZ kutengera mfundo ya immunoassay ya enzyme immunoassay.Zitsime za microtiter zimakutidwa ndi antigen yolumikizidwa ndi BSA.AOZ mu zitsanzo amapikisana ndi antigen yokutidwa pa mbale ya microtiter ya antibody yomwe yawonjezeredwa.Pambuyo pakuwonjezera kwa enzyme conjugate, gawo lapansi la chromogenic limagwiritsidwa ntchito ndipo chizindikirocho chimayesedwa ndi spectrophotometer.Mayamwidwe ake ndi osagwirizana ndi kuchuluka kwa AOZ pachitsanzocho.

1. 3.Mapulogalamu

Zida izi zitha kugwiritsidwa ntchito pakuwunika kuchuluka komanso kuwongolera kwa zotsalira za AOZ mkatianima tissues(minofu, chiwindi ndi zina), uchi.

1. 4.Zosiyanasiyana

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.Zofunika

5.1Zida

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

----Zida zoyanika ndi nayitrogeni

----Homogenizer /stomacher

--- Shaker

---- Chosakaniza cha Vortex

--Chinthu chapakati

----Kuwerengera bwino (kulowetsa: 0.01g)

-Pipette yomaliza maphunziro: 10ml

----Babu la rabara

---- Botolo la Volumetric: 100ml

---- Botolo lagalasi: 10ml

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

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

250ul-multipipette

5.2Ma reagents

----Ethyl acetate (AR)

----n-hexane (kapena n-heptane) (AR)

---- Dipotassium hydrogen phosphate trihydrate

(K₂HPO₄.3H₂O) (AR)

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

------Methanol

----Sodium hydroxide (NaOH, AR)

----Madzi osungunuka

1. 6.Zida Zamagetsi

l Microtiter mbale yokutidwa ndi antigen, 96 zitsime

L njira Standard (6 mabotolo, 1ml/botolo)

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

l Kuwongolera kokhazikika: (1ml/botolo)…….100ppb

l enzyme yokhazikika conjugate 1ml........kapu yofiira

l Enzyme conjugate diluents 10ml ………..wobiriwira kapu

l Substrate A 7ml…………………………..…..kapu yoyera

l Gawo B 7ml……………………………….…..kapu yofiyira

l Stop solution 7ml………………………………kapu yachikasu

L 20 × anaikira kusamba njira 40ml

……………………………………………………

L 2 × anaikira m'zigawo njira 60ml….buluu kapu

l 2-Nitrobenzaldehyde 15.1mg………………kapu yoyera

1. 7.Kukonzekera kwa Reagents

Yankho 1: zotumphukira reagent:

Onjezani 10ml ya methanol ku botolo lokhala ndi 2-Nitrobenzaldehyde ndikusungunula.(pa ndende ya 10mM).

Yankho 2Mtengo: 0.1MK₂HPO₄yankho:

Kulemera 22.8g K₂HPO₄.3H₂O mpaka 1L madzi opangidwa kuti asungunuke.

Yankho 3: 1M HCl yankho

Sungunulani 8.3ml Concentrated hydrochloric acid ndi madzi deionized 100ml.

Yankho 4:1M NaOH yankho

Sungunulani 4g NaOH ndi 100ml deionized madzi.

Solution 5: njira yothetsera:

Sungunulani 2 × moyikira m'zigawo njira ndi madzi deionized mu chiŵerengero cha 1: 1.Njirayi ikhoza kusungidwa kwa 1month pa 4 ℃, yomwe idzagwiritsidwe ntchito pochotsa zitsanzo.

Yankho 6: wash solution:

Sungunulani 20 × concentrated kusamba njira ndi madzi deionized mu voliyumu mlingo wa 1:19, amene adzagwiritsidwa ntchito kutsuka mbale.Njira yothetsera izi imatha kusungidwa kwa mwezi umodzi pa 4 ℃.

1. 8.Zokonzekera Zitsanzo

8.1Zindikirani ndi kusamala musanagwire ntchito:

a) Chonde gwiritsani ntchito maupangiri amodzi pakuyesa, ndikusintha malangizowo mukamayamwa ma reagent osiyanasiyana.

b) Onetsetsani kuti zida zonse zoyesera ndi zoyera.

c) zotumphukira zotumphukira zimatha kusungidwa pa 2-8 ℃ kwa miyezi itatu;

d) Mankhwala a HCl amatha kusungidwa kutentha kwa miyezi itatu;

e) Njira ya NaOH ikhoza kusungidwa kwa miyezi itatu kutentha;

f) Sungani zitsanzo zosasamalidwa pozizira (-20 ℃);

g) Zitsanzo zothandizidwa zitha kusungidwa kwa 24h pa 2-8 ℃ mumdima.

8.2 Zitsanzo za minofu ya nyama ndi chiwindi:

---- Homogenize zitsanzo ndi homogenizer;

----Kulemera kwa 1.0 ± 0.05g kwa chitsanzo cha minofu ya homogenized kukhala 50ml polystyrene centrifuge chubu.Onjezani madzi opangidwa ndi 4ml, 0.5ml 1M HCl solution ndi 100ul derivative reagent (onani yankho 1).Gwirani kwa 2min.

---- Yalirani pa 37℃ usiku wonse (pafupifupi 16h);

---- Onjezani 5ml 0.1MK₂HPO₄ (yankho2), 0.4ml 1M NaOH (yankho4) ndi 5ml ethyl acetate.Kugwedeza mwamphamvu kwa 30s;

---- Centrifuge kutentha (20-25 ℃) kwa 10min, osachepera 3000g;

---- Tengani 2.5ml ya gawo lamphamvu kwambiri lachilengedwe mu chubu lagalasi loyera la 10ml, louma ndi mpweya wa nayitrogeni wa 50-60℃ kapena evaporator yozungulira;

---- Sungunulani zouma zotsalira ndi 1ml n-hexane (kapena n- heptane), vortex kwa 30s, onjezani 1ml m'zigawo zowonjezera (yankho5), vortex 1min, sakanizani kwathunthu.

---- Centrifuge kutentha kwapakati (20-25^oC) kwa 5min, osachepera 3000g;

---- Chotsani gawo lamphamvu lachilengedwe;Tengani 50μl ya gawo lamadzi la gawo lapansi kuti muyese.

8.4 Uchi

----kulemera 1.0±0.05g wa uchi wa homogenized mu chubu cha 50ml polystyrene centrifuge;

----Onjezani madzi opangidwa ndi 4ml, 0.5ml 1M HCl (yankho3) ndi 100μl zotumphukira zotumphukira (yankho1);gwedezani kwathunthu kwa 2min;

----Yalirani pa 37℃ usiku wonse (pafupifupi 16h);

----Onjezani 5ml 0.1MK₂HPO₄ (yankho2), 0.4ml 1M NaOH (yankho4) ndi 5ml ethyl acetate, gwedezani mwamphamvu kwa 30s;

----Centrifuge pa kutentha (20-25 ℃) kwa 10min, osachepera 3000g;

----Tengani 2.5ml ya gawo lamphamvu kwambiri la organic mu chubu lagalasi loyera la 10ml, louma ndi mpweya wa nayitrogeni wa 50-60℃ kapena evaporator yozungulira;

----Sungani zouma zotsalira ndi 1ml n-hexane (kapena n- heptane), vortex kwa 30s, onjezerani 1ml m'zigawo zosakaniza (yankho5), vortex 1min, sakanizani kwathunthu.

----Centrifuge pa kutentha kwapakati (20-25^oC) kwa 10min, osachepera 3000g;

---- Chotsani gawo lamphamvu lachilengedwe;Tengani 50μl ya gawo lamadzi la gawo lapansi kuti muyese.

1. 9 .Njira yoyesera

9.1Zindikirani musanayese

9.1.1 Onetsetsani kuti ma reagents ndi ma microwell onse ali pamalo otentha (20-25 ℃).

9.1.2 Bwezerani zotsalira zonse ku 2-8 ℃ mukangogwiritsa ntchito.

9.1.3 Kutsuka ma microwell molondola ndi gawo lofunikira pakuyesa;ndiye chinthu chofunikira kwambiri pakupanganso kusanthula kwa ELISA.

9.1.4 Pewani kuyatsa ndikuphimba tinthu tating'onoting'ono tomwe timayatsa.

9.2 Njira Zoyesera

9.2.1 Tulutsani ma reagents onse kutentha (20-25 ℃) kwa mphindi zopitilira 30, homogenize musanagwiritse ntchito.

9.2.2 Chotsani ma microwell ofunikira ndikubweza ena onse mu chikwama cha zip-lock pa 2-8 ℃ nthawi yomweyo.

9.2.3 Njira yosambitsira moyikirapo komanso njira yothira mozama iyenera kutenthedwa kuti ikhale yotentha musanayambe ntchito.

9.2.4Nambala:Kuwerengera malo aliwonse a microwell ndi miyezo yonse ndi zitsanzo ziyenera kuyendetsedwa mobwerezabwereza.Lembani miyeso ndi malo a zitsanzo.

9.2.5Kuchepetsa kwa concentrated antibody solution: chepetsani njira ya enzyme yokhazikika ndi enzyme conjugate diluents mu chiŵerengero cha voliyumu ya 1:10 (1 fold concentrated enzyme solution: 10 folds enzyme conjugate diluents).

9.2.6Onjezani njira yokhazikika / zitsanzo ndi yankho la enzyme conjugate: onjezani 50µl ya yankho lokhazikika kapena zitsanzo zokonzedwa kuzitsime zofananira, onjezani 50µl enzyme conjugate solution.Sakanizani mofatsa pogwedeza mbaleyo pamanja ndikuumirira kwa 30min pa 25 ℃ ndi chophimba.

9.2.7Sambani: Chotsani chivundikiro pang'onopang'ono ndikutsanulira madziwo m'zitsime ndikutsuka ma microwells ndi 250µl wosungunuka wosungunuka (yankho6) pa interval ya 10s kwa 4-5 zina.Yankhani madzi otsalawo ndi pepala loyamwa (mpweya wotsalawo ukhoza kuthetsedwa ndi nsonga yosagwiritsidwa ntchito).

9.2.8Mitundu: Onjezani njira ya 50µl ya gawo lapansi A ndi 50ul ya gawo B pa chitsime chilichonse.Sakanizani mofatsa pogwedeza mbale pamanja ndikuumirira kwa mphindi 15 pa 25 ℃ ndi chophimba (onani 12.8).

9.2.11Yesani: Onjezani 50µl yankho loyimitsa pachitsime chilichonse.Sakanizani pang'onopang'ono pogwedeza mbale pamanja ndi kuyeza kuyamwa pa 450nm (Muyezedwe ndi mafunde apawiri a 450/630nm. Werengani zotsatira mkati mwa 5min mutawonjezera njira yoyimitsa.)

10 Zotsatira

10.1Peresenti absorbance

Ziwerengero zamtengo wapatali zomwe zimatengedwa pamiyeso ndi zitsanzo zimagawidwa ndi mtengo wamtengo wapatali wamtundu woyamba (zero standard) ndikuchulukitsa ndi 100%.Muyezo wa zero umapangidwa kukhala wofanana ndi 100% ndipo milingo ya kuyamwa imatchulidwa mwamaperesenti.

=

B --absorbance muyezo (kapena chitsanzo)

B0 --absorbance zero muyezo

10.2Mapindikira Okhazikika

----Kujambulira mulingo wokhotakhota: Tengani kuchuluka kwa milingo ngati y-axis, semi logarithmic ya kuchuluka kwa njira yokhazikika ya AOZ (ppb) ngati x-axis.

----Kuphatikizika kwa AOZ kwachitsanzo chilichonse (ppb), chomwe chitha kuwerengedwa kuchokera pa curve ya calibration, kumachulukitsidwa ndi gawo lofananira lachitsanzo chilichonse chotsatiridwa, ndipo kuchuluka kwenikweni kwa zitsanzo kumapezedwa.

Chonde zindikirani:

Mapulogalamu apadera apangidwa pofuna kuchepetsa deta, zomwe zingaperekedwe popempha.

Dilution factor………………………………………………………2

10.Sensitivity, kulondola ndi kulondola

Kumverera: 0.025ppb

Malire ozindikira:

Minofu (minofu, chiwindi)…………………………….0.1ppb

Honey------------------------------------------------ -0.1ppb

Kulondola:

Minofu ya nyama (minofu ndi chiwindi)……………………100±20%

Honey……………………………………………………………………………………………………………. 100±20%

Kulondola:CV ya zida za ELISA ndi zosakwana 10%.

11.Zindikirani

12.1 Zomwe zikutanthawuza zazitsulo zowonongeka zomwe zimaperekedwa kwa miyezo ndi zitsanzo zidzachepetsedwa ngati ma reagents ndi zitsanzo sizinakonzedwe kutentha kwa chipinda (20-25 ℃).

12.2 Musalole ma microwells kuti awume pakati pa masitepe kuti mupewe kuberekana kosatheka ndikugwiritsanso ntchito sitepe yotsatira mukangogwira chosungira ma microwells.

12.3 Gwirani reagent iliyonse mofatsa musanagwiritse ntchito.

12.4 Sungani khungu lanu kutali ndi njira yoyimitsa chifukwa ndi 0.5MH₂SO₄yankho.

12.5 Musagwiritse ntchito zida zakale.Osasinthanitsa ma reagents amagulu osiyanasiyana, apo ayi zitha kutsitsa chidwi.

12.6 Malo osungira: Sungani zida za ELISA pa 2-8 ℃, musazimitse.Sindikizani mbale zopumira, Pewani kuwala kwadzuwa nthawi zonse mu incubation.Kuphimba mbale microtiter tikulimbikitsidwa.

12.7 Njira yothetsera vutoli iyenera kusiyidwa ngati isintha mitundu.Ma reagents amatha kuwonongeka ngati mphamvu yakuya (450/630nm) ya zero muyezo ndi yochepera 0.5 (A450nm<0.5).

12.8 Maonekedwe amtundu amafunikira 15min pambuyo powonjezera yankho la gawo lapansi;Mukhoza kutalikitsa nthawi yoyamwitsa mpaka 20min kapena kuposerapo ngati mtunduwo ndi wopepuka kwambiri kuti udziwike., osapitirira 25min. M'malo mwake, fupikitsani nthawi yoyenera.

12.9 The mulingo woyenera kwambiri kutentha ndi 25 ℃.Kutentha kwakukulu kapena kutsika kumayambitsa kusintha kwa chidwi ndi kuyamwa.

12.Malo osungira ndi nthawi yosungira

Malo osungira: 2-8 ℃.

Nthawi yosungira: 12months.