umkhiqizo

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.