chigadzirwa

1. 1.Background

Nitrofurans inogadzirwa ne-broad-spectrum antibiotics, iyo inowanzoshandiswa mukugadzirwa kwemhuka kune yayo yakanaka antibacterial uye pharmacokinetic properties.Vakanga vashandiswa zvakare sevanosimudzira kukura munguruve, huku uye kugadzirwa kwemvura.Muzvidzidzo zvenguva refu nemhuka dzemurabhoritari zvakaratidza kuti mishonga yevabereki uye metabolites yavo yakaratidza carcinogenic uye mutagenic maitiro.Izvi zvakakonzera kurambidzwa kwe nitrofuran pakurapa mhuka dzinoshandiswa kugadzira chikafu.Mishonga ye nitrofuran furaltadone, nitrofurantoin uye nitrofurazone yakarambidzwa kushandiswa mukugadzirwa kwemhuka yezvokudya muEU mu1993, uye kushandiswa kwefurazolidone kwakarambidzwa muna 1995.

Kuongororwa kwe nitrofuran zvinodhaka zvakasara zvinoda kubva pakuonekwa kwetishu yakasungwa metabolites yemishonga yevabereki nitrofuran.Sezvo mishonga yevabereki inokurumidza kukurumidza kushandiswa, uye mishonga inosungirirwa nitrofuran metabolites ichachengetedza kwenguva yakareba, iyi metabolites inoshandiswa sechinangwa mukuonekwa kwekushungurudzwa kwe nitrofurans, iyo inosanganisira Furazolidone metabolite (AOZ), Furaltadone metabolite (AMOZ). ), Nitrofurantoin metabolite (AHD) uye Nitrofurazone metabolite (SEM).

AOZ-zvisaririra zvinotemerwa zvakanyanya neLC-MS kana LC-MS/MS.Enzyme immunoassays, kana ichienzaniswa nechromatographic nzira, inoratidza zvakati wandei zvakanakira kunzwa, yekuona muganho, tekinoroji michina uye nguva inodiwa.(Nguva inodhura: 45min)

1. 2.Test Principle

Iyi ELISA kit yakagadzirirwa kuona AOZ zvichibva pamusimboti weiyo indirect-competitive enzyme immunoassay.Iyo microtiter matsime akaputirwa nekubata BSA-yakabatana antigen.AOZ mumuenzaniso inokwikwidza neantigen yakavharwa pa microtiter plate yeantibody yakawedzerwa.Mushure mekuwedzera kwe enzyme conjugate, chromogenic substrate inoshandiswa uye chiratidzo chinoyerwa ne spectrophotometer.Kunyura kwacho kunopesana nechikamu cheAOZ mumuenzaniso.

1. 3.Applications

Iyi kit inogona kushandiswa muhuwandu uye hwemhando yekuongorora kweAOZ yakasara mukatianima tissues(tsandanyama, chiropa etc), huchi.

1. 4.Cross-reactions

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.Zvinhu Zvinodiwa

5.1Equipments

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

----Rotary evaporator kana nitrogen yekuomesa zviridzwa

----Homogenizer /stomacher

-----Shaker

---- Vortex musanganisi

----Centrifuge

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

---- Pipette akapedza kudzidza: 10ml

----Rubber pipette girobhu

----Volumetric flask: 100ml

---- Girazi furasiki: 10ml

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

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

250ul-multipipette

5.2Reagents

----Ethyl acetate (AR)

----n-hexane (kana n-heptane) (AR)

---- Dipotassium hydrogen phosphate trihydrate

(K₂HPO₄.3H₂O) (AR)

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

-----Methanol

----Sodium hydroxide (NaOH, AR)

-----Deionized mvura

1. 6.Kit Zvikamu

l Microtiter ndiro yakavharwa neantigen, 96 matsime

l Yakajairwa mhinduro (6 mabhodhoro, 1ml / bhodhoro)

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

l Spiking standard control : (1ml/bhodhoro)........100ppb

l Concentrated enzyme conjugate 1ml........red cap

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

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

l Substrate B 7ml…………………………………….…..kapu tsvuku

l Stop solution 7ml………………………………yellow cap

l 20 × yakanyanyisa kushambidza mhinduro 40ml

……………………………………………..transparent cap

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

l 2-Nitrobenzaldehyde 15.1mg………………chivharo chichena

1. 7.Reagents Kugadzirira

Solution 1: derivative reagent:

Wedzera 10ml yemethanol kubhodhoro rine 2-Nitrobenzaldehyde uye nyungudika.(pahuwandu hwe10mM).

Solution 2: 0.1MK₂HPO₄mhinduro:

Huremu 22.8g K₂HPO₄.3H₂O kune 1L yemvura yakasvibiswa kuti inyunguduke.

Solution 3: 1M HCl mhinduro

Dissolve 8.3ml Concentrated hydrochloric acid nemvura yakasvibiswa kusvika ku100ml.

Mhinduro 4:1M NaOH mhinduro

Dissolve 4g NaOH ne 100ml deionized mvura.

Solution 5: mhinduro yekubvisa:

Dilute 2 × concentrated extraction solution nemvura yakasvibiswa muchiyero che1: 1.Mhinduro iyi inogona kuchengetedzwa kwe1month pa4 ℃, inozoshandiswa kuburitsa samples.

Solution 6: wash solution:

Dilute iyo 20 × yakagadziriswa yekushambidza mhinduro nemvura yakasvibiswa muhuwandu hwehuwandu hwe1:19, iyo ichashandiswa kushambidza mahwendefa.Iyi mhinduro yakasvibiswa inogona kuchengetedzwa kwemwedzi 1 pa4 ℃.

1. 8.Kugadzirira Mienzaniso

8.1Chiziviso uye zvidziviriro usati washanda:

a) Ndokumbira ushandise matipi e-imwe-muyedzo, uye shandura matipi paunenge uchitora reagent yakasiyana.

b) Ita shuwa kuti zvese zviridzwa zvekuyedza zvakachena.

c) derivative reagent inogona kuchengetedzwa pa2-8 ℃ kwemwedzi mitatu;

d) Mushonga weHCl unogona kuchengetedzwa mukamuri tembiricha kwemwedzi mitatu;

e) Mhinduro yeNaOH inogona kuchengetedzwa kwemwedzi mitatu patembiricha yekamuri;

f) Chengeta masampuli asina kubatwa muchando (-20 ℃);

g) Sampuli dzakarapwa dzinogona kuchengetedzwa kwe24h pa2-8 ℃ murima.

8.2 Zvinyama zvemhuka uye zviropa samples:

---- Homogenize masampuli ne homogenizer;

----Huremu 1.0±0.05g hwesample yetishu yehomogenized mu50ml polystyrene centrifuge chubhu.Wedzera 4ml deionized mvura, 0.5ml 1M HCl solution uye 100ul derivative reagent (ona mhinduro 1).Shake iyo kwe2min.

---- Ramba pa37 ℃ usiku hwose ( dzinenge 16h);

---- Wedzera 5ml 0.1MK₂HPO₄ (mhinduro2), 0.4ml 1M NaOH (mhinduro4) uye 5ml ethyl acetate.Kuzunungusa kunotyisa kwe30s;

---- Centrifuge pakamuri tembiricha (20-25 ℃) nokuti 10min, kanenge 3000g;

---- Tora 2.5ml yechikamu chepamusoro-soro muchubhu yegirazi yakachena 10ml, yakaoma ne50-60℃ nitrogen gasi kana rotary evaporator;

---- Dissolve iyo yakaoma yasara ne 1ml n-hexane (kana n- heptane), vortex ye30s, wedzera 1ml yekubvisa mhinduro (mhinduro5), vortex 1min, sanganisa zvachose.

---- Centrifuge pakamuri tembiricha (20-25^oC) ye5min, kanenge 3000g;

---- Bvisa iyo supernatant organic chikamu;tora 50μl yechikamu chemvura ye substrate kuti uedze.

8.4 Huchi

----kuyera 1.0±0.05g yehuchi huchi sampuli mu50ml polystyrene centrifuge chubhu;

----Wedzera 4ml mvura yakasvibiswa, 0.5ml 1M HCl (mhinduro3) uye 100μl yakatorwa reagent (mhinduro1);zunza zvachose 2min;

----Imbai pa37℃ usiku humwe (anenge 16h);

----Wedzera 5ml 0.1MK₂HPO₄ (mhinduro2), 0.4ml 1M NaOH (mhinduro4) uye 5ml ethyl acetate, kuzunungusa kunotyisa kwe30s;

----Centrifuge pakamuri tembiricha (20-25℃) nokuda 10min, kanenge 3000g;

----Tora 2.5ml ye supernatant organic phase mu 10ml yakachena girazi chubhu, yakaoma ne 50-60 ℃ nitrogen gasi kana rotary evaporator;

----Dingura iyo yakaoma yasara ne 1ml n-hexane (kana n- heptane), vortex ye30s, wedzera 1ml yekubvisa mhinduro (mhinduro5), vortex 1min, sanganisa zvachose.

----Centrifuge pakupisa kwekamuri (20-25^oC) kwe10min, kanenge 3000g;

----Bvisa iyo supernatant organic chikamu;tora 50μl yechikamu chemvura ye substrate kuti uedze.

1. 9.Assay process

9.1Cherechedza usati waongororwa

9.1.1 Iva nechokwadi chekuti mareagents nemamicrowell ese ari pakamuri tembiricha (20-25℃).

9.1.2 Dzosera mamwe ese asara ku2-8℃ pakarepo mushure mekushandisa.

9.1.3 Kuwacha ma microwells nemazvo inhanho yakakosha muchirongwa chekuongorora;ndicho chinhu chakakosha pakuberekazve kweiyo ELISA kuongororwa.

9.1.4 Nzvenga chiedza uye vhara mamicrowell panguva yekuisa.

9.2 Matanho Ekuongorora

9.2.1 Bvisa mareagents ese kunze kwekamuri tembiricha (20-25 ℃) kweanopfuura 30min, homogenize usati washandisa.

9.2.2 Budisa ma microwell anodiwa kunze uye wodzosera asara mubhegi rezip-lock pa2-8℃ nekukasika.

9.2.3 Mushonga wekugezesa wakanyunguduka uye mushonga wakanyunguduka wekubvisa unofanirwa kudziyirwa kuti ugare uchidziya tembiricha usati washandiswa.

9.2.4Nhamba:Yakaverengerwa ese ma microwell zvinzvimbo uye ese mazinga uye masampuli anofanirwa kuitiswa muduplicate.Rekodha zviyero uye masampuli nzvimbo.

9.2.5Dilution ye concentrated antibody solution: deredza iyo concentrated enzyme solution ne enzyme conjugate diluents muhuwandu hwehuwandu hwe1: 10 (1 fold concentrated enzyme solution: 10 folds enzyme conjugate diluents).

9.2.6Wedzera yakajairwa mhinduro / sampuli uye enzyme conjugate mhinduro: wedzera 50µl yeyakajairwa mhinduro kana sampu yakagadzirwa kumatsime anoenderana, wedzera 50µl enzyme conjugate mhinduro.Sanganisa zvinyoro nyoro nekuzunza ndiro nemaoko uye pinza kwe30min pa25℃ nebutiro.

9.2.7Geza: Bvisa chivharo zvinyoronyoro wodira mvura kubva mumatsime uye sukurudza ma microwells ne 250µl diluted wash solution (mhinduro6) panguva ye10s ye4-5 nguva.Sveta mvura yasara nepepa rinonyudza (yakasara mhepo bubble inogona kubviswa nemuromo usina kushandiswa).

9.2.8Coloration: Wedzera 50µl substrate solution A uye 50ul substrate solution B patsime rega rega.Sanganisa zvinyoro nyoro nekuzunguza ndiro nemaoko uye pinza kwemaminetsi gumi nemashanu pa25 ℃ nekavha (ona 12.8).

9.2.11Kuyera: Wedzera 50µl mhinduro yekumisa patsime rega rega.Sanganisa zvinyoro nyoro nekuzunguza ndiro nemaoko uye kuyera kunyura pa450nm (Yakarakadza kuyerwa nedual-wavelength ye450/630nm. Verenga mhedzisiro mukati me5min mushure mekuwedzera kwekumisa mhinduro.)

10 Results

10.1Percentage absorbance

Izvo zvinorehwa maitiro ezviyero zvekutora zvakawanikwa zvezviyero uye sampuli zvinopatsanurwa nehuwandu hwekutora hwekutanga (zero standard) uye inowedzerwa ne100%.Iyo zero chiyero inoitwa yakaenzana ne100% uye iyo yekutora tsika inotorwa muzvikamu.

=

B --absorbance standard (kana sampuli)

B0 --absorbance zero standard

10.2Standard Curve

----Kudhirowa curve yakajairwa: Tora kukosha kwekunyudza kwemazinga se y-axis, semi logarithmic yeconcentration yeAOZ standard solution (ppb) se x-axis.

----Iyo yeAOZ yemuenzaniso wega wega (ppb), iyo inogona kuverengwa kubva pakuenzanisa curve, inowedzerwa neinopindirana dilution factor yemuenzaniso wega wega unoteverwa, uye chiyero chaicho chemuenzaniso chinowanikwa.

Tapota cherechedzai:

Yakakosha software yakagadziridzwa kune ese kuderedza data, iyo inogona kupihwa pakukumbira.

Dilution factor……………………………………………………2

10.Sensitivity, kururama uye nemazvo

Sensitivity: 0.025ppb

Muganho wekuona:

Matumbu (tsandanyama, chiropa)…………………………….0.1ppb

Huchi------------------------------------------------ -0.1ppb

Kururama:

Zvinyama zvemhuka (tsandanyama nechiropa)……………………100±20%

Uchi ………………………………………………….. 100±20%

Precision:CV ye ELISA kit iri pasi pe10%.

11.Cherechedzai

12.1 Izvo zvinorehwa maitiro ehutano hwekutora hunowanikwa kune zviyero uye sampuli dzichaderedzwa kana reagents uye sampuli dzisina kugadziriswa kukushisa kwekamuri (20-25 ℃).

12.2 Usabvumira mamicrowell kuti aome pakati pematanho kudzivirira kusabudirira kuberekana uye shandisa danho rinotevera nekukurumidza mushure mekutapa iyo microwells inobata.

12.3 Zunza reagent yega yega zvinyoro nyoro usati washandisa.

12.4 Chengetedza ganda rako kure nemhinduro yekumira nekuti ndiyo 0.5MH₂SO₄mhinduro.

12.5 Usashandise kits dzechinyakare.Usatsinhane mareagents emabhechi akasiyana, kana zvikasadaro anodonha senitivity.

12.6 Mamiriro ekuchengetedza: Chengetedza ELISA kits pa2-8 ℃, usaomesa.Simbisa zororo microwell mahwendefa, Dzivisa chiedza chezuva chakatwasuka panguva dzese incubations.Kuvhara mahwendefa e microtiter kunokurudzirwa.

12.7 Substrate mhinduro inofanira kusiiwa kana ichichinja mavara.Iyo reagents inogona kuderedzwa kana iyo absorbance value (450/630nm) yezero standard iri pasi pe0.5 (A450nm <0.5).

12.8 Iyo coloration reaction inoda 15min mushure mekuwedzera kwe substrate mhinduro;Iwe unogona kuwedzera nguva yekudzivirira kusvika ku20min kana kupfuura kana ruvara rwakanyanya kujeka kuti usarudze., usambopfuura 25min. Pane zvinopesana, pfupisa nguva yekudzivirira zvakanaka.

12.9 Iyo yakakwana yekuita tembiricha i25 ℃.Kupisa kwepamusoro kana kuderera kunotungamira kune shanduko yekunzwa uye kunyura maitiro.

12.Mamiriro ekuchengetedza uye nguva yekuchengetedza

Kuchengetedza mamiriro: 2-8 ℃.

Nguva yekuchengetedza: 12months.