Competitive Enzyme Immunoassay Kit for Quantitative Analysis of Tylosin
Mpikisano wa Enzyme Immunoassay Kit wa
Quantitative Analysis ofTylosin
1. Mbiri
Tylosinndi macrolide antibiotic, omwe amagwiritsidwa ntchito makamaka ngati antibacterial ndi anti-mycoplasma.Ma MRL okhwima sanakhazikitsidwe popeza mankhwalawa angayambitse zotsatira zoyipa m'magulu ena.
Chida ichi ndi chinthu chatsopano chotengera ukadaulo wa ELISA, womwe ndi wachangu, wosavuta, wolondola komanso wozindikira poyerekeza ndi kusanthula kwa zida zomwe zimafunikira maola 1.5 pakuchita ntchito imodzi, zitha kuchepetsa kulakwitsa kwantchito komanso kulimba kwa ntchito.
2. Mfundo Yoyesera
Chida ichi chimachokera paukadaulo wa ELISA wosalunjika.Zitsime za microtiter zimakutidwa ndi antigen yolumikizana.Zotsalira za tylosin pachitsanzo zimapikisana ndi antigen yokutidwa pa mbale ya microtiter ya antibody.Pambuyo powonjezera enzyme yotchedwa anti-antibody, gawo lapansi la TMB limagwiritsidwa ntchito kusonyeza mtundu.Kusapezeka kwa chitsanzocho kumagwirizana molakwika ndi tylosin amakhala mmenemo, pambuyo poyerekezera ndi Standard Curve, kuchulukitsidwa ndi dilution factor, tylosin residue quantity mu chitsanzo ikhoza kuwerengedwa.
3. Mapulogalamu
Zidazi zitha kugwiritsidwa ntchito pakuwunika kwachulukidwe komanso koyenera kwa zotsalira za tylosin mu minofu yanyama (nkhuku, nkhumba, bakha) ndi mkaka, uchi, dzira, ndi zina zambiri.
4. Kusagwirizana
Tylosin…………………………………………………………..100%
Tilmicosin …………………………………………………<2%
5. Zinthu Zofunika
5.1 Zipangizo:
---- Microtiter mbale spectrophotometer (450nm/630nm)
----Zida zoyanika ndi nayitrogeni
-- homogenizer
--- Shaker
--Chinthu chapakati
----Kuwerengera bwino (kulowetsa: 0.01g)
-Pipette yomaliza maphunziro: 10ml
----Babu la rabara
---- Botolo la Volumetric: 10ml
----Machubu a polystyrene centrifuge: 50ml
----Micropipettes: 20-200ml, 100-1000ml
250ml-multipipette
5.2 Reagents:
----Sodium hydroxide (NaOH, AR)
Sodium bicarbonate (NaHCO3,AR)
- Sodium carbonate (NaCO3, AR)
----Trichloroacetic acid (AR)
---- Acetonitrile (AR)
----Ethyl acetate (AR)
┅┅n-Hexane (AR)
----Madzi osungunuka
6. Zigawo za Kit
l mbale ya Microtiter yokhala ndi zitsime 96 zokutidwa ndi antigen
L njira Standard (5 mabotolo, 1ml/botolo)
0ppb, 0.5ppb, 1.5ppb, 4.5ppb, 13.5ppb
l Spiking muyezo ulamuliro: (1ml/botolo)1 ppm
l Enzyme conjugate 1ml……………..…..…kapu yofiira
l Antibody solution 7ml ……………….……kapu yobiriwira
l Solution A 7ml…………………….….……kapu yoyera
l Solution B 7ml…………………………………..kapu yofiira
l Stop solution 7ml.……………….……….kapu yachikasu
L 20 × anaikira kusamba njira 40ml
…………………………………………….. transparent cap
L 4 × anaikira m'zigawo njira 50ml
……………………………………………………….blue cap
7. Kukonzekera kwa Reagents:
Yankho 1:0.1mol/L NaOH yankho
Yesani 0.4g NaOH mpaka 100ml madzi opangidwa ndi deionized ndikusakaniza kwathunthu.
Solution 2: 1mol/L NaOH yankho
Yesani 4g NaOH mpaka 100ml madzi opangidwa ndi deionized ndikusakaniza kwathunthu.
Solution 3: Mchere wa carbonate buffer
Yankho1: 0.2M PB
Sungunulani 51.6g wa Na2HPO4· 12H2O, 8.7g ya NaH2PO4· 2H2O ndi madzi deionized ndi kuchepetsa 1000ml.
Yankho2: Njira yochotsera
Sungunulani 2 × concentrated m'zigawo njira ndi madzi deionized mu voliyumu chiŵerengero cha 1: 1 (mwachitsanzo 10ml ya 2× m'zigawo njira + 10ml wa madzi deionized), zomwe zingagwiritsidwe ntchito pochotsa zitsanzo,njira iyi akhoza kusungidwa pa 4 ℃ kwa 1 mwezi.
Yankho3: Sambani njira
Sungunulani 20 × concentrated kutsuka njira ndi madzi deionized mu voliyumu chiŵerengero cha 1:19 (mwachitsanzo 5ml ya 20×kusamba njira + 95ml yamadzi opangidwa ndi deionized), zomwe zidzagwiritsidwe ntchito kutsuka mbale.Njirayi ikhoza kusungidwa pa 4 ℃ kwa mwezi umodzi.
8. Zitsanzo Zokonzekera
8.1 Chidziwitso ndi kusamala musanagwire ntchito:
(a) Chonde gwiritsani ntchito malangizo amodzi poyesa, ndikusintha malangizowo mukamamwa reagent yosiyana.
(b) Onetsetsani kuti zida zonse ndi zoyera.
(c) Sungani minyewa yoziziritsa kukhosi.
(d) Chitsanzo chokonzekera chiyenera kugwiritsidwa ntchito poyesa nthawi imodzi.
8.2 Nyama ya nyama (nkhuku, nkhumba, ndi zina)
---- Homogenize chitsanzo ndi homogenizer;
----Tengani 2.0±0.05g ya homogenate mu chubu cha 50ml polystyrene centrifuge;onjezani 2ml ya 0.2M PB (yankho1) , gwedezani kuti musungunuke, kenaka yikani 8ml ya ethyl acetate ndikugwedezani mwamphamvu kwa 3min;
----Centrifuge yolekanitsa: 3000g / kutentha kozungulira / 5min.
----- Tumizani 4ml ya gawo lamphamvu kwambiri la organic mu chubu lagalasi la 10ml, louma ndi madzi osambira a 50-60 ℃ pansi pa mtsinje wa nayitrogeni;
----Sungani zowuma zotsalira ndi 1ml wa n-hexane, vortex kwa 30s kuti zisungunuke, kenaka onjezerani 1ml ya m'zigawo zosakaniza (yankho2), vortex kwa 1min.centrifuge kupatukana: 3000g / yozungulira kutentha / 5min
---- Chotsani gawo lamphamvu la n-hexane;tengani 50μl ya gawo lapansi lamadzi kuti muyese.
Dilution factor: 1
8.2 Mkaka
----Tengani 100μl ya mkaka wosaphika, sakanizani ndi 900μl ya m'zigawo (yankho2), ndi kusakaniza kwathunthu.
----Tengani 50μl ya yankho lomwe lakonzedwa kuti muyese.
Dilution factor: 10
9. Njira yoyesera
9.1 Zindikirani musanayese
9.1.1Onetsetsani kuti ma reagents ndi ma microwell onse ali kutentha (20-25 ℃).
9.1.2Bweretsani ma reagents ena onse ku 2-8℃mwamsanga pambuyo ntchito.
9.1.3Kutsuka ma microwells molondola ndi gawo lofunikira pakuyesa;ndiye chinthu chofunikira kwambiri pakupanganso kusanthula kwa ELISA.
9.1.4 Achotsa kuwala ndikuphimba ma microwells panthawi yoyamwitsa.
9.2 Njira Zoyesera
9.2.1 Tulutsani ma reagents onse kutentha (20-25 ℃) kwa mphindi zopitilira 30, gwedezani pang'ono musanagwiritse ntchito.
9.2.2 Chotsani ma microwell ofunikira ndikubweza ena onse mu chikwama cha zip-lock pa 2-8 ℃ nthawi yomweyo.
9.2.3 Njira yotsuka yotsuka iyenera kutenthedwa kuti ikhale yotentha kwambiri musanagwiritse ntchito.
9.2.4Nambala:Kuwerengera malo aliwonse a microwell ndi miyezo yonse ndi zitsanzo ziyenera kuyendetsedwa mobwerezabwereza.Lembani miyeso ndi malo a zitsanzo.
9.2.5Add standard solution/chitsanzo ndi antibody solution: Onjezani 50µl ya njira yokhazikika ((zida zoperekedwa)) kapena chitsanzo chokonzekera ku zitsime zofananira.Onjezani 50µl ya antibody solution (zida zoperekedwa).Sakanizani mofatsa pogwedeza mbaleyo pamanja ndikuumirira kwa 30min pa 37 ℃ ndi chophimba.
9.2.6Sambani: Chotsani chivundikirocho pang'onopang'ono ndikuyeretsa madziwo m'zitsime ndikutsuka ma microwells ndi 250µl diluted wash solution (yankho3) pa interval ya 10s kwa 4-5times.Yankhani madzi otsalawo ndi pepala loyamwa (mpweya wotsalawo ukhoza kuthetsedwa ndi nsonga yosagwiritsidwa ntchito).
9.2.7Onjezerani ma enzyme conjugateOnjezani 100ml ya enzyme conjugate solution (zida zoperekedwa) pachitsime chilichonse, sakanizani mofatsa ndikuulira kwa 30min pa 37 ℃ ndi chophimba.Bwerezaninso sitepe yochapa kachiwiri.
9.2.8Mitundu: Onjezani 50µl ya yankho A(zida zoperekedwa) ndi 50µl ya yankho B (zida zoperekedwa) ku chitsime chilichonse.Sakanizani mofatsa ndikuumirira kwa 15min pa 37 ℃ ndi chophimba.
9.2.9Yesani: Onjezani 50µl ya njira yoyimitsa (zida zoperekedwa) ku chitsime chilichonse.Sakanizani pang'onopang'ono ndikuyesa kuyamwa pa 450nm (Ndiyezo muyeso ndi wavelength wapawiri wa 450/630nm. Werengani zotsatira mkati mwa 5min mutawonjezera njira yoyimitsa).
10. Zotsatira
10.1 Peresenti ya absorbance
Ziwerengero zamtengo wapatali zazitsulo zomwe zimatengedwa pamiyeso ndi zitsanzo zimagawidwa ndi kulowetsedwa kwa mlingo woyamba (zero standard) ndikuchulukitsa ndi 100%.Muyezo wa zero umapangidwa kukhala wofanana ndi 100% ndipo milingo ya kuyamwa imatchulidwa mwamaperesenti.
B
Absorbance (%) = —— × 100%
B0
B --absorbance muyezo (kapena chitsanzo)
B0 --absorbance zero muyezo
10.2 Njira Yokhazikika
----Kujambula mulingo wokhotakhota: Tengani kuchuluka kwa milingo ngati y-axis, semi logarithmic ya concentration ya tylosin standards solution (ppb) ngati x-axis.
----Kuphatikizika kwa tylosin kwa sampuli iliyonse (ppb), komwe kumatha kuwerengedwa kuchokera pamapindikira owongolera, kumachulukitsidwa ndi gawo lofananira la Dilution lachitsanzo chilichonse chotsatiridwa, ndipo kuchuluka kwenikweni kwa zitsanzo kumapezedwa.
Chonde zindikirani:
pulogalamu yapadera yapangidwa kuti ifufuze deta, yomwe ingaperekedwe popempha.
11. Kumverera, kulondola ndi kulondola
Kumverera kwa Mayeso:1.5ppb
Malire ozindikira:
Nyama ya nyama ………………………………………………… 1.5ppb Mkaka…………………………………………………………..15ppb Kulondola:
Minofu ya nyama …………………………………………….80±15%
Mkaka…………………………………………………….80±10%
Kulondola:
Kusiyanasiyana kwa zida za ELISA ndizochepera 10%.
12. Zindikirani
12.1 Zomwe zikutanthawuza zazitsulo zomwe zimatengedwa pamiyeso ndi zitsanzo zidzachepetsedwa ngati ma reagents ndi zitsanzo sizinakonzedwe kutentha kwa chipinda (20-25 ℃).
12.2 Musalole ma microwells kuti awume pakati pa masitepe kuti mupewe kuberekana kosatheka ndikugwiritsanso ntchito sitepe yotsatira mukangogwira chosungira ma microwells.
12.3 Gwirani reagent iliyonse mofatsa musanagwiritse ntchito.
12.4 Sungani khungu lanu kutali ndi njira yoyimitsa chifukwa ndi 0.5MH2SO4yankho.
12.5 Musagwiritse ntchito zida zakale.Osasinthanitsa ma reagents amagulu osiyanasiyana, apo ayi zitha kutsitsa chidwi.
12.6 Sungani zida za ELISA pa 2-8 ℃, osazizira.Sindikizani mbale zopumira, Pewani kuwala kwadzuwa nthawi zonse mu incubation.Kuphimba mbale microtiter tikulimbikitsidwa.
12.7 Njira yothetsera vutoli iyenera kusiyidwa ngati isintha mitundu.Ma reagents atha kukhala oyipa ngati mphamvu yakuya (450/630nm) ya zero muyezo ndiyochepera 0.5 (A450nm<0.5).
12.8 Maonekedwe amtundu amafunikira 15min mutatha kuwonjezera yankho la A ndi yankho B. Ndipo mutha kutalikitsa nthawi ya makulitsidwe mpaka 20min kapena kupitilira apo ngati mtunduwo ndi wopepuka kwambiri kuti udziwike.Osapitirira 30min, m'malo mwake, kufupikitsa nthawi yoyamwitsa bwino.
12.9 The mulingo woyenera kwambiri kutentha ndi 37 ℃.Kutentha kwakukulu kapena kutsika kumayambitsa kusintha kwa chidwi ndi kuyamwa.
13. Kusungirako
Malo osungira: 2-8 ℃.
Nthawi yosungira: miyezi 12.
