umkhiqizo

Ikhithi Yokuncintisana Ye-Enzyme Immunoassay Yokuhlaziya Okuningi kwe-Flumequine

Incazelo emfushane:

I-Flumequine iyilungu le-quinolone elwa namagciwane, esetshenziswa njenge-anti-infective ebaluleke kakhulu emkhiqizweni wezilwane kanye nowasemanzini ngenxa yobubanzi bayo, ukusebenza kahle okuphezulu, ubuthi obuphansi kanye nokungena kwezicubu eziqinile.Ibuye isetshenziselwe ukwelapha izifo, ukuvimbela kanye nokukhuthaza ukukhula.Ngoba kungaholela ekuphikisweni kwezidakamizwa kanye nokuba khona kwe-carcinogenicity, umkhawulo ophezulu lapho ngaphakathi kwezicubu zezilwane unqunywe e-EU, eJapane (umkhawulo ophezulu ngu-100ppb e-EU).

Njengamanje, i-spectrofluorometer, i-ELISA kanye ne-HPLC izindlela eziyinhloko zokuthola izinsalela ze-flumequine, futhi i-ELISA ibe yindlela evamile yokuzwela okuphezulu nokusebenza okulula.


Imininingwane Yomkhiqizo

Omaka bomkhiqizo

Isimiso Sokuhlola

Le khithi isuselwe kubuchwepheshe obungaqondile bokuncintisana be-ELISA.Imithombo ye-microtiter ihlanganiswe ne-antigen ehlangene.I-Flumequineinsalela kusampula iqhudelana ne-antigen embozwe epuleti le-microtiter le-antibody.Ngemva kokwengezwa kwe-enzyme ebizwa ngokuthi i-anti-antibody, i-TMB substrate isetshenziselwa ukukhombisa umbala.Ukungabi khona kwesampula kuhlobene kabi nokuhlala kwe-tetracycline kuyo, ngemva kokuqhathanisa ne-Standard Curve, ephindaphindwa ngokuphindaphinda kwe-dilution, inani lensalela ye-Flumequine kusampula lingabalwa.

Izinhlelo zokusebenza

Le khithi ingasetshenziswa ekuhlaziyeni inani kanye nekhwalithi yensalela ye-flumequine ojuni .

Ukusabela okuphambene

I-Flumequine ………………………………………………… 100%

 

Izinto Ezidingekayo

Izisetshenziswa

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

┅┅Homogenizer noma isisu

┅┅I-Shaker

┅┅I-Vortex mixer

┅┅Centrifuge

┅┅Ibhalansi yokuhlaziya (i-inductance: 0.01g)

┅┅I-pipette ethweswe iziqu: 15ml

┅┅I-rubber pipette bulb

┅┅Polystyrene Centrifuge tube: 15ml, 50ml

┅┅Ithubhu yokuhlola yengilazi:10ml

┅┅Amapayipi amancane: 20ml-200ml, 100ml -10000ml,

250ml - i-multipipette

Ama-reagents

┅┅n-hexane(AR)

┅┅Methylene chloride(AR)

┅┅I-Acetonitrile(AR)

┅┅Amanzi akhishwe ngaphandle

-----I-hydrochloric acid egxilile(AR)

 

Izingxenye Zekhithi

● Ipuleti le-Microtiter elinemithombo engu-96 embozwe nge-antigen

● Izisombululo ezijwayelekile(6 amabhodlela×1ml/ibhodlela)

0ppb, 0.3ppb, 1.2ppb, 4.8ppb, 19.2ppb, 76.8ppb

● Izinga eliphezulu lokulawula izinga:(1ml/ibhodlela)

………………………………………………………….100ppb

● I-enzyme conjugate 12ml……………………………… ikepisi elibomvu

● Isixazululo se-antibody 7ml …………..……..….…..ikepisi eliluhlaza

● Isixazululo A 7ml……..……………………..………..ikepisi elimhlophe

● Isixazululo B 7ml …………….……………..………… ikepisi elibomvu

● Isixazululo sokumisa 7ml ……………………..………ikepisi eliphuzi

● 20XIsixazululo sokugeza esigxilile esingu-40ml

…………………………………………..…..i-cap esobala

●2X Isixazululo Sokukhipha 50ml……………………… ikepisi eliluhlaza okwesibhakabhaka

 

Ukulungiswa kwama-reagents

7.1 Isampula yoju

Isixazululo 1 : 0.2 M Isixazululo se-Hydrochloric acid

Isisindo esingu-41.5ml I-Hydrochloric acid egxilile, ihlanjululwe ngamanzi e-deionized kuya ku-500 ml.

Isixazululo 2: Isixazululo sokugeza

Nciphisa isixazululo sokugeza esigxilile ngamanzi akhishwe ngevolumu isilinganiso esingu-1:19, esizosetshenziselwa ukugeza amapuleti.ikhambi elihlanjululwe lingagcinwa ku-4 ℃ inyanga engu-1.

Isixazululo3: isixazululo sokukhipha

Nciphisa isixazululo sokukhipha okugxilile esingu-2× ngamanzi akhishwe ngevolumu ngesilinganiso sevolumu engu-1:1 (noma kuya ngesidingo), esizosetshenziselwa ukukhipha isampula.Lesi sixazululo esihlanjululwe singalondolozwa inyanga engu-1 ku-4 ℃.

Amalungiselelo Esampula

8.1 Izaziso nezixwayiso zabasebenzisi ngaphambi kokusebenza

(a) Sicela usebenzise amathiphu aphuma kanye kunqubo yokuhlola, futhi ushintshe amathiphu lapho uthatha i-reagent ehlukile.

(b) Qiniseka ukuthi wonke amathuluzi okuhlola ahlanzekile, ngaphandle kwalokho kuzosebenza umphumela wokuhlola.

8.2Isampula yoju

-----Kala isampula yoju engu-2g±0.05g ibe yishubhu ye-polystyrene centrifuge engu-50ml,

-----Engeza u-2ml 0.2 M Isixazululo se-Hydrochloric acid (Isixazululo 1), i-vortex ukuze uyixube ngokuphelele, bese wengeza u-8ml we-methylene chloride, unyakazise nge-shaker imizuzu emi-5 ukuze uncibilike ngokuphelele;

-----Centrifuge 10 min, okungenani 3000g ekamelweni lokushisa (20-25℃);

-----Susa isigaba esinamandla amakhulu, thatha u-2 ml wesisombululo se-substrate se-organic eshubhu lengilazi elingu-10 ml. sula indawo engaphansi ngaphansi kokugeza kwamanzi okugeleza kwe-Nitrogen (50-60℃)

-----Engeza 1 ml n-hexane,vortex for 30s, bese wengeza 1ml isixazululo sokukhipha(isixazululo 3) ,vortex futhi imizuzu engu-1.I-Centrifuge ye-5min, okungenani i-3000g ekamelweni lokushisa (20-25℃);

-----Susa isigaba esinamandla amakhulu, thatha u-50ml ukuze uhlole;

9. Inqubo yokuhlola

9.1 Qaphela ngaphambi kokuhlolwa

9.1.1 Qinisekisa ukuthi zonke izinto ezisebenza ngogesi nama-microwell konke kusezingeni lokushisa elilingana negumbi (20-25℃).

9.1.2 Buyisela wonke amanye ama-reagents ku-2-8℃ ngokushesha ngemva kokusetshenziswa.

9.1.3 Ukugeza ama-microwell ngendlela efanele kuyisinyathelo esibalulekile ohlelweni lokuhlola;kuyisici esibalulekile ekuphindaphindweni kokuhlaziywa kwe-ELISA.

9.1.4 Gwema ukukhanya futhi umboze ama-microwell ngesikhathi sokufukamela.

9.2 Izinyathelo Zokuhlola

9.2.1 Khipha wonke ama-reagents ekamelweni lokushisa (20-25℃) ngaphezu kwama-30min, i-homogenize ngaphambi kokusetshenziswa.

9.2.2 Khipha ama-microwell adingekayo bese ubuyisela amanye esikhwameni se-zip-lock ku-2-8℃ ngokushesha.

9.2.3 Ingxube yokugeza ehlanjululwe kufanele ifudunyezwe kabusha ukuze ibe sezingeni lokushisa elilingana negumbi ngaphambi kokusetshenziswa.

9.2.4Inombolo:Faka izinombolo kuzo zonke izindawo ze-microwell futhi wonke amazinga namasampuli kufanele asetshenziswe ngokuphindiwe.Rekhoda amazinga kanye nezikhundla zamasampuli.

9.2.5Engeza isisombululo/isampula ejwayelekile:Engeza u-50 µl wesixazululo esijwayelekile noma isampula elilungisiwe emithonjeni ehambisanayo.Engeza isisombululo se-antibody esingu-50µl.Hlanganisa ngobumnene ngokunyakazisa ipuleti ngesandla bese ufukamela imizuzu engama-30 ku-25℃ ngekhava.

9.2.6Geza:Khipha ikhava ngobumnene bese uhlanza uketshezi emithonjeni bese uhlanza ama-microwells ngesisombululo sokugeza esihlanjululwe esingu-250µl (isixazululo 2) ngesikhawu se-10s izikhathi ezi-4-5.Gcoba amanzi asele ngephepha elimuncayo (ibhamuza lomoya elisele lingaqedwa ngethiphu elingasetshenzisiwe).

9.2.8.Ukuhlanganiswa kwe-enzyme:Faka i-enzyme conjugate solution 100ml emthonjeni ngamunye, Xuba ngobumnene ngokunyakazisa ipuleti ngesandla bese ufukamela imizuzu engama-30 ku-25℃ ngekhava.Phinda isinyathelo sokugeza futhi.

9.2.8Umbala:Engeza isixazululo esingu-50µl A kanye nekhambi elingu-50µl B emthonjeni ngamunye.Xuba ngobumnene ngokunyakazisa ipuleti ngesandla bese ufukamela imizuzu eyi-15 ku-25℃ ngesembozo (bona 12.8).

9.2.9Kala:Engeza isixazululo sokumisa esingu-50µl emthonjeni ngamunye.Xuba kahle ngokunyakazisa ipuleti ngokwenza bese ukala ukumunca ku-450nm endaweni engenalutho yomoya (Kuyaphakanyiswa isilinganiso esinobude obumbili begagasi obungu-450/630nm. Funda umphumela phakathi kwemizuzu emi-5 ngemva kokwengeza isixazululo sokumisa. ) (Singakwazi futhi ukukala ngokubona ngaphandle kwesixazululo sokumisa ngamafuphi ithuluzi le-ELIASA)

Imiphumela

10.1 Iphesenti lokumunca

Amanani amaphakathi amavelu okumunca atholwe kumazinga kanye namasampuli ahlukaniswa ngevelu yokumunca yezinga lokuqala (izinga elinguziro) futhi liphindwe ngo-100%.Izinga elinguziro lenziwa lalingana no-100% futhi amanani okumunca acashunwe ngamaphesenti.

I-Absorbance (%) = B/B0 × 100%

B ——izinga le-absorbance (noma isampula)

B0 ——absorbance zero standard

10.2 Ijika Elijwayelekile

Ukuze udwebe ijika elijwayelekile: Thatha inani lokumunca lamazinga njenge-y-axis, i-semi logarithmic ye-concentration ye-flumequine standards solution (ppb) njenge-x-eksisi.

--- Thei-flumequineukugxila kwesampula ngayinye (ppb), engafundwa kwijika lokulinganisa, kuphindwa ngokuphindaphinda okuhambisanayo kwesampula ngayinye elandelwayo, futhi ukugxiliswa kwangempela kwesampula kuyatholakala.

Ukuze kuncishiswe idatha ye-ELISA kits, isofthiwe ekhethekile iye yathuthukiswa, enganikezwa ngesicelo.

11. Ukuzwela, ukunemba nokunemba

Ukuzwela kokuhlola:0.3ppb

Uju lwesampula lokuhlanjululwa kwesici: 2

Umkhawulo wokutholwa

Isampula yoju----------------------------------------------- -1ppb

Ukunemba

Isampula yoju -------------------------------------------- 90±20 %

Ukunemba

I-Coefficient ehlukile yekhithi ye-ELISA ingaphansi kuka-10%.

12. Qaphela

12.1 Amanani amaphakathi wamanani okumunca atholwe kumazinga kanye namasampula azoncishiswa uma ama-reagents namasampuli engalawulwanga kumazinga okushisa egumbi (20-25℃).

12.2 Ungavumeli ama-microwell ome phakathi kwezinyathelo ukugwema ukuphindaphinda okungaphumelelanga futhi usebenzise isinyathelo esilandelayo ngokushesha ngemva kokuthepha isibambi sama-microwells.

12.3.I-Homogenize i-reagent ngayinye ngaphambi kokusebenzisa.

12.4.Gcina isikhumba sakho kude nesixazululo sokumisa ngoba yi-2M H2SO4isisombululo.

12.5 Ungawasebenzisi amakhithi asephelelwe yisikhathi.Ungashintshi ama-reagents amaqoqo ahlukene, ngoba azokwehlisa ukuzwela.

12.6 Isimo sesitoreji:

Gcina amakhithi e-ELISA ku-2-8℃, ungafrizi.Vala amapuleti amancane okuphumula Gwema ukukhanya kwelanga okuqondile phakathi nawo wonke ama-incubation.Kunconywa ukumboza amapuleti e-microtiter.

12.7 Izinkomba zokungahambi kahle kwama-reagents:

Isixazululo se-substrate kufanele sishiywe uma sishintsha imibala.

Ama-reagents angase abe mabi uma inani lokumunca (450/630nm) lezinga elinguziro lingaphansi kuka-0.5 (A450nm<0.5).

12.8 Ukusabela kombala kudinga imizuzu engu-15 ngemva kokwengeza Isixazululo A kanye Nesixazululo B. Futhi ungakwazi ukwelula ubude besikhathi sokufukamela ukusuka kumaminithi angu-20 kuye kwangaphezulu uma umbala ulula kakhulu ukuthi unganqunywa.Ungalokothi udlule i-25min, Ngokuphambene nalokho, finyeza isikhathi sokufukamela ngendlela efanele.

12.9 Izinga lokushisa elilungile lokusabela ngu-25℃.Izinga lokushisa eliphakeme noma eliphansi lizoholela ekushintsheni kokuzwela kanye namanani okumunca.

13. Isitoreji

Isimo sesitoreji: 2-8℃.

Isikhathi sokugcina: izinyanga eziyi-12.


  • Okwedlule:
  • Olandelayo:

  • Bhala umlayezo wakho lapha futhi usithumelele wona