imveliso

1. Imvelaphi

Tylosinsisibulali-zintsholongwane se-macrolide, esisetyenziswa ikakhulu njenge-antibacterial kunye ne-anti-mycoplasma.Ii-MRLs ezingqongqo azisekiwe njengoko eli chiza linokukhokelela kwisiphumo esibi kakhulu kumaqela athile.

Le khithi yimveliso entsha esekwe kwitekhnoloji ye-ELISA, ekhawulezayo, elula, echanekileyo kwaye enovelwano xa kuthelekiswa nohlalutyo oluqhelekileyo lwesixhobo kwaye ifuna iiyure eziyi-1.5 kuphela kumsebenzi omnye, inokunciphisa kakhulu impazamo yokusebenza kunye nokuqina komsebenzi.

2. Umgaqo-siseko woVavanyo

Le khithi isekelwe kwitekhnoloji ye-ELISA engathanga ngqo.Imithombo ye-microtiter ifakwe kwi-antigen yokudibanisa.Intsalela yeTylosin kwisampulu ikhuphisana ne-antigen egqunywe 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-tylosin ehlala kuyo, emva kokuthelekisa kunye ne-Standard Curve, iphindwe yi-dilution factor, ubuninzi be-tylosin residue kwisampuli ingabalwa.

3. Izicelo

Le kiti ingasetyenziselwa uhlalutyo lobungakanani kunye nomgangatho wentsalela ye-tylosin kwizicubu zezilwanyana (inkukhu, ingulube, idada) kunye nobisi, ubusi, iqanda, njl.

4. Iimpendulo ezahlukeneyo

Tylosin……………………………………………………..100%

Tilmicosin…………………………………………………<2%

5. Izinto ezifunekayo

5.1 Izixhobo:

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

----Umphunga ojikelezayo okanye izixhobo zokomisa initrogen

-------homogenizer

-----Shaker

-----Iziko

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

----Uthweswe isidanga: 10ml

----Ibhalbhu yerabha yepayipi yerabha

-----Iflaski yeVolumetric: 10ml

----Iityhubhu ze-Polystyrene centrifuge: 50ml

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

250ml-multipipette

5.2 Iiarhente:

----Isodium hydroxide (NaOH, AR)

-------Sodium bicarbonate (NaHCO_3,AR)

---- I-Sodium carbonate (NaCO₃, AR)

-----Trichloroacetic acid (AR)

---- Acetonitrile (AR)

----Ethyl acetate (AR)

┅┅n-Hexane (AR)

---- Amanzi agayiweyo

6. Amacandelo eKit

l I-Microtiter plate enamaqula angama-96 aqatywe nge-antigen

l Izisombululo ezisemgangathweni (iibhotile ezi-5, 1ml/ibhotile)

0ppb, 0.5ppb, 1.5ppb, 4.5ppb, 13.5ppb

l Ulawulo oluqhelekileyo lwe-Spiking: (1ml/ibhotile)1ppm

l I-Enzyme conjugate 1ml……………..…..…ikepusi ebomvu

l Isisombululo se-Antibody 7ml……………….……ikepusi eluhlaza

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

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

l Misa isisombululo 7ml.…………….……….yellow cap

l 20 × isisombululo sokuhlamba esigxininisiweyo 40ml

…………………………………………………

l 4 × isisombululo esigxininisiweyo sokukhupha i-50ml

………………………………………………….ikepusi eluhlaza

7. Ukulungiselela iiReagents:

Isisombululo 1:I-0.1mol / L isisombululo se-NaOH

Ubunzima be-0.4g NaOH ukuya kwi-100ml yamanzi e-deionized kwaye udibanise ngokupheleleyo.

Isisombululo 2: I-1mol / L isisombululo se-NaOH

Ubunzima be-4g NaOH ukuya kwi-100ml yamanzi e-deionized kwaye udibanise ngokupheleleyo.

Isisombululo 3: Ityuwa ye-carbonate buffer

Isisombululo1: 0.2M PB

Nciphisa i-51.6g yeNa₂HPO₄·12H₂O, 8.7g ye-NaH₂PO₄·2H₂O ngamanzi adibeneyo kwaye ahlambulule kwi-1000ml.

Isisombululo2: Isisombululo sokukhupha

Nciphisa isisombululo se-2 × esigxininisiweyo sokukhupha ngamanzi adibeneyo kumlinganiselo wevolumu ye-1: 1 (umz. i-10ml ye-2× isisombululo sokukhupha + 10ml yamanzi adibeneyo), eya kusetyenziselwa utsalo lwesampulu,esi sisombululo sinokugcinwa kwi-4 ℃ kwinyanga eyi-1.

Isisombululo3: Hlamba isisombululo

Nciphisa i-20 × isisombululo sokuhlamba esigxininisiweyo kunye namanzi adibeneyo kumlinganiselo wevolumu ye-1:19 (umz. i-5ml ye-20×isisombululo sokuhlamba + 95ml yamanzi adiyiniweyo), eza kusetyenziselwa ukuhlamba iipleyiti.Esi sisombululo sinokugcinwa kwi-4 ℃ kwinyanga enye.

8. Amalungiselelo Isampulu

8.1 Isaziso kunye nezilumkiso phambi kokusebenza:

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

(b) Qinisekisa ukuba zonke izixhobo zokusebenza zicocekile.

(c) Gcina isampulu yethishu kumkhenkce.

(d) Isampulu elungisiweyo kufuneka isetyenziselwe uvavanyo kwangoko.

8.2 Izicubu zezilwanyana (inkukhu, inyama yehagu, njl.njl)

-----Homogenize isampuli nge-homogenizer;

----Thatha i-2.0±0.05g ye-homogenate kwi-50ml ye-polystyrene centrifuge tube;yongeza i-2ml ye-0.2M PB (isisombululo1) , xubha ukunyibilika, uze ungeze i-8ml ye-ethyl acetate kwaye ugubungele ngokukrakra kwi-3min;

----I-Centrifuge yokwahlula: 3000g / ubushushu be-ambient / 5min.

----Ukutshintshela i-4ml yesigaba se-organic supernatant kwi-10ml yeglasi ityhubhu, yomile kunye ne-50-60℃ yokuhlamba amanzi phantsi komjelo wegesi ye-nitrogen;

----Nyibilikisa intsalela eyomileyo nge-1ml ye-n-hexane, i-vortex ye-30s ukunyibilika, kwaye wongeze i-1ml yesisombululo sokukhupha (isisombululo2), i-vortex ye-1min.i-centrifuge yokwahlula: 3000g / ubushushu be-ambient / 5min

----Susa inqanaba le-n-hexane elinamandla;thatha i-50μl yesigaba sokumanzisa engaphantsi ukuze uvavanye.

Imeko yokunciphisa: 1

8.2 Ubisi

----Thatha i-100μl yesampuli yobisi ekrwada, xuba ne-900μl yesisombululo sokukhupha (isisombululo2), kwaye udibanise ngokupheleleyo.

----Thatha i-50μl yesisombululo esilungisiweyo sovavanyo.

Imeko yokunciphisa: 10

9. Inkqubo yovavanyo

9.1 Qaphela phambi kovavanyo

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

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

9.1.3Ukuhlamba i-microwells ngokuchanekileyo linyathelo elibalulekileyo kwinkqubo yokuvavanya;yeyona nto ibalulekileyo ekuveliseni kwakhona uhlalutyo lwe-ELISA.

9.1.4 Avala isibane kwaye ugqume iimicrowells ngexesha lokufukamela.

9.2 Amanyathelo oVavanyo

9.2.1 Zikhuphe zonke ii-reagents kwiqondo lobushushu begumbi (20-25℃) ngaphezulu kwe-30min, vuthulula ngobunono phambi kokuba uyisebenzise.

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:Inani lezithuba zemicrowell zonke kunye nemigangatho kunye neesampulu kufuneka ziqhutywe ngokuphindwe kabini.Rekhoda imigangatho kunye neendawo zeesampulu.

9.2.5Add isisombululo esisemgangathweni/isampuli kunye nesisombululo se-antibody: Yongeza i-50µl yesisombululo esisemgangathweni ((ikhithi inikezelwe)) okanye isampuli elungisiweyo ukuya kumaqula ahambelanayo.Yongeza i-50µl yesisombululo se-antibody (ikhithi inikezelwe).Xuba ngobunono ngokushukumisa ipleyiti ngesandla kwaye ufukame i-30min kwi-37℃ ngegquma.

9.2.6Hlamba: Susa isigqumathelo ngobunono kwaye ucoce ulwelo oluphuma equleni kwaye uhlambulule iimicrowells nge 250µl isisombululo sokuhlamba esixutyiweyo (isisombululo3) ngexesha le-10 ye-4-5 amaxesha.Funxa amanzi ashiyekileyo ngephepha elifunxayo (iqamza lomoya eliseleyo linokupheliswa ngencam engasetyenziswanga).

9.2.7Yongeza i-enzyme conjugateYongeza i-100ml yesisombululo se-enzyme conjugate (ikhithi inikezelwe) kwiqula ngalinye, xuba ngobunono kwaye ufukame i-30min nge-37℃ ngegquma.Phinda inyathelo lokuhlamba kwakhona.

9.2.8UmbalaYongeza i-50µl yesisombululo A(ikhithi inikezelwe) kunye ne-50µl yesisombululo B (ikhithi inikezelwe) kwiqula ngalinye.Xuba ngobunono kwaye ufukamele i-15min kwi-37℃ ngegquma.

9.2.9Umlinganiselo: Yongeza i-50µl yesisombululo sokuyeka (ikhithi inikezelwe) kwiqula ngalinye.Xuba ngobunono kwaye ulinganise i-absorbence kwi-450nm (Kucetyiswa umlinganiselo kunye ne-double-wavelength ye-450 / 630nm. Funda umphumo ngaphakathi kwe-5min emva kokufaka isisombululo sokumisa).

10. Iziphumo

10.1 Ipesenti yokuthatha

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

B

Ukungabikho (%) = —— × 100%

B0

B ——umgangatho wokungabikho (okanye isampuli)

B0 —-absorbance zero umgangatho

10.2 Ijika eliMgangatho

----Ukuzoba igophe elisemgangathweni: Thatha ixabiso lokufunxa lemigangatho njenge-y-axis, isemi logarithmic yoxinaniso lwesisombululo semigangatho yetylosin (ppb) njenge-x-axis.

----I-tylosin concentration yesampuli nganye (ppb), enokufundwa kwi-curve yokulinganisa, iphindwe nge-Dilution factor ehambelanayo yesampuli nganye elandelwayo, kwaye i-concentration yangempela yesampuli ifunyenwe.

Nceda uqaphele:

isoftware ekhethekileyo iye yaphuhliswa ukwenzela uhlalutyo lwedatha, enokuthi ibonelelwe ngesicelo.

11. Uvakalelo, ukuchaneka nokuchaneka

Uvakalelo loVavanyo:1.5ppb

Umda wokufunyanwa:

Ithishu yezilwanyana ………………………………………………1.5ppb Ubisi…………………………………………………………..15ppb Ukuchaneka:

Ithishu yezilwanyana……………………………………………80±15%

Ubisi………………………………………………..……80±10%

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 ukuba zome phakathi kwamanyathelo ukuphepha ukuphindaphinda okungaphumelelanga kwaye usebenzise inyathelo elilandelayo ngokukhawuleza emva kokucofa isibambi se-microwells.

12.3 Vuthulula i-reagent nganye ngobunono phambi kokusetyenziswa.

12.4 Gcina ulusu lwakho kude nesisombululo sokumisa kuba yi-0.5MH₂SO₄isisombululo.

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

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

12.7 Isisombululo seSubstrate kufuneka siyekwe ukuba sijika imibala.I-reagents ingaba yimbi ukuba ixabiso le-absorbeance (450/630nm) lomgangatho we-zero lingaphantsi kwe-0.5 (A450nm <0.5).

12.8 Ukusabela kombala kufuna i-15min emva kokongezwa kwesisombululo A kunye nesisombululo B. Kwaye unokwandisa ixesha lokufukamela ukuya kwi-20min okanye ngaphezulu ukuba umbala ukhaphukhaphu kakhulu ukuba ungamiselwa.Ungaze udlule kwi-30min, ngokuchaseneyo, nciphisa ixesha lokufukamela ngokufanelekileyo.

12.9 Elona qondo lobushushu lokusabela yi-37℃.Ubushushu obuphezulu okanye obuphantsi buya kukhokelela ekutshintsheni kobuntununtunu kunye nexabiso lokuthatha.

13. Ugcino

Imeko yokugcina: 2-8℃.

Ixesha lokugcinwa: iinyanga ezili-12.