imveliso

Umgaqo woVavanyo

Le khithi isekelwe kwitekhnoloji ye-ELISA engathanga ngqo.Imithombo ye-microtiter ifakwe kwi-antigen yokudibanisa.FlumequineIntsalela kwisampulu ikhuphisana ne-antigen eqatywe kwipleyiti ye-microtiter ye-antibody.Emva kokongezwa kwe-enzyme ebizwa ngokuba yi-anti-antibody, i-substrate ye-TMB isetyenziselwa ukubonisa umbala.I-Absorbance yesampuli inxulumene kakubi ne-tetracycline ehlala kuyo, emva kokuthelekisa kunye ne-Standard Curve, iphindwe nge-dilution multiple, i-Flumequine i-residue quantity kwisampuli ingabalwa.

Usetyenziso

Le khithi ingasetyenziselwa uhlalutyo lobungakanani kunye nomgangatho wentsalela yeflumequine kubusi.

Iimpendulo ezinqamlezayo

I-Flumequine ………………………………………………… 100%

Izinto Eziyimfuneko

Izixhobo

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

┅┅Homogenizer okanye isisu

┅┅Ishaker

┅┅Umxube weVortex

┅┅Iziko

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

┅┅Ipipette ethweswe isidanga: 15ml

┅┅Ibhalbhu yerabha yepayipi yerabha

┅┅Polystyrene Centrifuge tube: 15ml, 50ml

┅┅Ityhubhu yovavanyo lweglasi:10ml

┅┅Micropipettes: 20ml-200ml, 100ml -10000ml,

250ml -i-multipipette

Iireagents

┅┅n-hexane(AR)

┅┅Methylene chloride(AR)

┅┅Acetonitrile(AR)

┅┅Amanzi adityanisiweyo

-----I-hydrochloric acid egxininisiweyo (AR)

Izixhobo zeKit

● Icwecwe leMicrotiter elinamaqula angama-96 aqatywe ngeantigen

● Izisombululo eziqhelekileyo (iibhotile ezi-6×1ml/ibhotile)

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

● Ulawulo olukumgangatho ophezulu woxinaniso:(1ml/ibhotile)

………………………………………………………….100ppb

● I-Enzyme conjugate 12ml…………………………... ikepusi ebomvu

● Isisombululo se-antibody 7ml …………..……..….…..ikepusi eluhlaza

● Isisombululo A 7ml……..……………………..………..ikepusi emhlophe

● Isisombululo B 7ml …………….……………..………… ikepusi ebomvu

● Misa isisombululo 7ml ……………………..…………ikepusi etyheli

● I-20XIsisombululo sokuhlamba esigxininisiweyo se-40ml

……………………………………………..…..icap ecacileyo

●2X Isisombululo sokukhupha 50ml…………………………

Ukulungiswa kweeReagents

7.1 Isampulu yobusi

Isisombululo 1 : 0.2 M Isisombululo se-Hydrochloric acid

Ubunzima 41.5ml I-Hydrochloric acid egxininisiweyo, ihlanjululwe ngamanzi adibeneyo ukuya kwi-500 ml.

Isisombululo 2: Hlamba isisombululo

Nciphisa isisombululo sokuhlamba esigxininisiweyo ngamanzi adiyiweyo kwi-volume ratio ye-1:19, eya kusetyenziselwa ukuhlamba iipleyiti.isisombululo esihlanjululweyo sinokugcinwa kwi-4 ℃ kwinyanga eyi-1.

Isisombululo3: isisombululo sokukhupha

Nciphisa i-2 × isisombululo sokutsalwa okugxininisiweyo ngamanzi adibeneyo kwi-volume ration ye-1: 1 (okanye ixhomekeke kwimfuno), eya kusetyenziselwa ukukhutshwa kwesampuli.Esi sisombululo sihlanjululweyo sinokugcinwa inyanga enye kwi-4℃.

Amalungiselelo Isampulu

8.1 Isaziso kunye nezilumkiso kubasebenzisi ngaphambi kokuba usebenze

(a) Nceda usebenzise iingcebiso zolunye kwinkqubo yovavanyo, kwaye utshintshe iingcebiso xa ufunxa i-reagent eyahlukileyo.

(b) Qinisekisa ukuba zonke izixhobo zovavanyo zicocekile , kungenjalo iya kuba nesiphumo sovavanyo.

8.2Isampulu yobusi

-----Wayisha i-2g ± 0.05g yesampuli yobusi kwi-50ml ye-polystyrene centrifuge tube,

-----Yongeza i-2ml 0.2 M isisombululo se-Hydrochloric acid (Isisombululo 1), i-vortex ukuyixuba ngokupheleleyo, uze udibanise i-8ml i-methylene chloride, xubha nge-shaker ye-5min ukuchithwa ngokupheleleyo;

-----I-Centrifuge ye-10 min, ubuncinane i-3000g kwindawo yokushisa (20-25℃);

-----Susa inqanaba eliphezulu, thatha i-2 ml ye-substrate yesisombululo se-organic kwi-tube yeglasi eyi-10 ml. yomisa i-substate phantsi kokuhlamba kwamanzi okuhamba kwe-Nitrogen (50-60℃)

-----Yongeza i-1 ml i-n-hexane,i-vortex ye-30s, emva koko wongeze isisombululo se-1ml sokukhupha (isisombululo sesi-3), i-vortex kwakhona nge-1min.I-Centrifuge ye-5min, ubuncinane i-3000g kwiqondo lokushisa (20-25℃);

-----Susa isigaba esiphezulu, thatha i-50ml yokuvavanya;

9. Inkqubo yovavanyo

9.1 Qaphela phambi kovavanyo

9.1.1 Qinisekisa ukuba zonke ii-reagents kunye ne-microwells zonke zikwiqondo lobushushu legumbi (20-25℃).

9.1.2 Buyisela zonke ezinye ii-reagents ku-2-8℃ ngokukhawuleza emva kokusetyenziswa.

9.1.3 Ukuhlamba iimicrowells ngokuchanekileyo linyathelo elibalulekileyo kwinkqubo yovavanyo;yinto ebalulekileyo yokuphindaphinda uhlalutyo lwe-ELISA.

9.1.4 Kuphephe ukukhanya kwaye wogqume iimicrowells ngexesha lokufukamela.

9.2 Amanyathelo oVavanyo

9.2.1 Thatha zonke ii-reagents kwiqondo lobushushu legumbi (20-25℃) ngaphezu kwe-30min, i-homogenize phambi kokusetyenziswa.

9.2.2 Khupha ii-microwells ezifunekayo kwaye ezinye uzibuyisele kwi-zip-lock bag ku-2-8℃ ngokukhawuleza.

9.2.3 Umxube wokuhlamba ohlanjululweyo kufuneka ufudunyezwe kwakhona ukuze ube kwiqondo lobushushu begumbi ngaphambi kokusetyenziswa.

9.2.4Inani:Faka iinombolo kwindawo nganye ye-microwell kwaye yonke imigangatho kunye neesampuli kufuneka ziqhutywe ngokuphindwe kabini.Rekhoda imigangatho kunye neendawo zeesampulu.

9.2.5Yongeza isisombululo esisemgangathweni/isampuli:Yongeza i-50 µl yesisombululo esisemgangathweni okanye isampulu elungisiweyo kumaqula ahambelanayo.Yongeza isisombululo se-antibody esiyi-50µl.Xuba ngobunono ngokushukumisa ipleyiti ngesandla kwaye ufukame i-30min kwi-25℃ ngegquma.

9.2.6Hlamba:Susa isigqumathelo ngobunono kwaye ucoce ulwelo oluphuma equleni kwaye uhlambulule imicrowells nge 250µl isisombululo sokuhlamba esixutyiweyo (isisombululo 2) ngekhefu le-10s kumaxesha ama-4-5.Funxa amanzi ashiyekileyo ngephepha elifunxayo (iqamza lomoya eliseleyo linokupheliswa ngencam engasetyenziswanga).

9.2.8.I-enzyme conjugate:Yongeza isisombululo se-enzyme ye-conjugate 100ml kwiqula ngalinye, Xuba ngobumnene ngokushukumisa ipleyiti ngesandla kwaye ufukamele i-30min kwi-25℃ nge-cover.Phinda inyathelo lokuhlamba kwakhona.

9.2.8Umbala:Yongeza i50µl isisombululo A kunye ne50µl isisombululo B kwiqula ngalinye.Xuba ngobunono ngokushukumisa ipleyiti ngesandla kwaye ufukamele i-15 min kwi-25℃ ngegquma (jonga i-12.8).

9.2.9Umlinganiselo:Yongeza 50µl isisombululo sokumisa kwiqula ngalinye.Xuba ngobunono ngokushukumisa ipleyiti ngesandla kwaye ulinganise ukufunxa kwi-450nm ngokuchasene nesithuba somoya (Kucetyiswa umlinganiselo wobude obubini be-450/630nm. Funda isiphumo kwimizuzu emi-5 emva kokongezwa kwesisombululo sokumisa. ) (Sinokulinganisa nangokubona ngaphandle kokumisa isisombululo ngokufutshane kwesixhobo se-ELIASA)

Iziphumo

10.1 Ipesenti yokuthatha

Amaxabiso aphakathi amaxabiso e-absorbence afunyenwe kwimigangatho kunye neesampuli zahlulwe ngexabiso lokuthatha umgangatho wokuqala (umgangatho we-zero) kwaye zanda nge-100%.Umgangatho we-zero wenziwa ulingane ne-100% kwaye amaxabiso e-absorpsor acatshulwe ngokweepesenti.

Ukungabikho (%) = B/B0 × 100%

B ——umgangatho wokungabikho (okanye isampuli)

B0 —-absorbance zero umgangatho

10.2 Ijika eliMgangatho

Ukuzoba igophe elisemgangathweni: Thatha ixabiso lokufunxa kwemigangatho njenge-y-axis, i-semi logarithmic yoxinaniso lwesisombululo semigangatho ye-flumequine (ppb) njenge-x-axis.

--- IflumequineUgxininiso lwesampulu nganye (ppb), enokuthi ifundwe kwi-curve yokulinganisa, iphindaphindwe ngokuphindaphindiweyo kwe-dilution yesampuli nganye elandelwayo, kwaye i-concentration yangempela yesampuli ifunyenwe.

Ukunciphisa idatha yeekhithi ze-ELISA, isoftware ekhethekileyo iye yaphuhliswa, enokunikezelwa ngesicelo.

11. Uvakalelo, ukuchaneka nokuchaneka

Uvakalelo loVavanyo:0.3ppb

Ubusi Isampulu yokuxutywa kwemeko: 2

Umda wokufunyanwa

Isampulu yobusi------------------------------------------------ -1ppb

Ukuchaneka

Isampulu yobusi --------------------------------------------- 90±20 %

Ukuchaneka

Ukwahluka komlinganiso wekhithi ye-ELISA ingaphantsi kwe-10%.

12. Qaphela

12.1 Amaxabiso aphakathi kwamaxabiso afunyenwe kwimigangatho kunye neesampuli ziya kuncitshiswa ukuba i-reagents kunye neesampuli azizange zilawulwe kwiqondo lokushisa kwegumbi (20-25℃).

12.2 Musa ukuvumela ii-microwells zome phakathi kwamanyathelo ukuphepha ukuphindaphinda okungaphumelelanga kwaye usebenzise inyathelo elilandelayo ngokukhawuleza emva kokucofa isibambi se-microwells.

12.3.Homogenize reagent nganye phambi kokuba usebenzise.

12.4.Gcina ulusu lwakho kude nesisombululo sokumisa kuba yi-2M H₂SO₄isisombululo.

12.5 Musa ukusebenzisa iikhithi eziphelelwe lixesha.Musa ukutshintshisa ii-reagents zeebhetshi ezahlukeneyo, kuba iya kuwisa ubuntununtunu.

12.6 Imeko yokugcina:

Gcina iikhithi ze-ELISA ku-2-8℃, musa ukuba ngumkhenkce.Zitywine iipleyiti ezincinci ze-microwell Kuphephe ukukhanya kwelanga ngexesha lonke lokufukamela.Kunconywa ukugubungela iiplati ze-microtiter.

12.7 Iimpawu zokungahambi kakuhle kwee-reagents:

Isisombululo se-substrate kufuneka sishiywe ukuba sijika imibala.

Ii-reagents zinokujika zibe zimbi ukuba ixabiso le-absorbeance (450/630nm) lomgangatho we-zero lingaphantsi kwe-0.5 (A450nm＜0.5).

12.8 Ukusabela kombala kufuna i-15min emva kokongeza iSicombululo A kunye neSicombululo B. Kwaye unokwandisa ixesha lokufukamela ukusuka kwi-20min ukuya ngaphezulu ukuba umbala ukhaphukhaphu kakhulu ukuba ungamiselwa.Ungaze udlule kwi-25min, Ngokuchasene noko, nciphisa ixesha lokufukamela ngokufanelekileyo.

12.9 Elona qondo lobushushu lokusabela liphezulu ngama-25℃.Ubushushu obuphezulu okanye obuphantsi buya kukhokelela ekutshintsheni kobuntununtunu kunye nexabiso lokuthatha.

13. Ugcino

Imeko yokugcina: 2-8℃.

Ixesha lokugcinwa: iinyanga ezili-12.