mankhwala

Mfundo Yoyesera

Chida ichi chimachokera paukadaulo wa ELISA wosalunjika.Zitsime za microtiter zimakutidwa ndi antigen yolumikizana.Flumequinezotsalira mu zitsanzo zimapikisana ndi antigen yokutidwa pa mbale ya microtiter ya antibody.Pambuyo powonjezera enzyme yotchedwa anti-antibody, gawo lapansi la TMB limagwiritsidwa ntchito kusonyeza mtundu.Kutaya kwachitsanzo kumagwirizana molakwika ndi tetracycline kukhala mmenemo, pambuyo poyerekeza ndi Standard Curve, kuchulukitsidwa ndi dilution angapo, Flumequine zotsalira kuchuluka mu chitsanzo akhoza kuwerengedwa.

Mapulogalamu

Zidazi zitha kugwiritsidwa ntchito pakuwunika kuchuluka komanso kuwongolera kwa zotsalira za flumequine mu uchi.

Zosiyanasiyana

Flumequine …………………………………………………… 100%

Zofunika

Zida

┅┅Microtiter mbale spectrophotometer (450nm/630nm)

┅┅Homogenizer kapena stomacher

┅┅Shaker

┅┅ Chosakaniza cha Vortex

┅┅Centrifuge

┅┅Kuwerengera bwino (kulowetsa: 0.01g)

┅┅Pipette yomaliza maphunziro: 15ml

┅┅Babu la rabara

┅┅Polystyrene Centrifuge chubu: 15ml, 50ml

┅┅Glasi yoyesera chubu: 10ml

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

250ml - multipipette

Ma reagents

┅┅n-hexane(AR)

┅┅Methylene chloride(AR)

┅┅Acetonitrile(AR)

┅┅Madzi osungunuka

-----Chidutswa cha hydrochloric acid(AR)

Zida Zamagetsi

● mbale ya Microtiter yokhala ndi zitsime 96 zokutidwa ndi antigen

● Njira zokhazikika (mabotolo 6×1ml/botolo)

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

● Kuwongolera kwapamwamba kwambiri: (1ml/botolo)

……………………………………………………………….100ppb

● Enzyme conjugate 12ml…………………………… kapu yofiira

● Antibody solution 7ml …………..……..….…..kapu yobiriwira

● Solution A 7ml……..……………………..………..kapu yoyera

● Solution B 7ml …………….……………..………… kapu yofiyira

● Stop solution 7ml ……………………..………kapu yachikasu

● 20XConcentrated kutsuka njira 40ml

………………………………………………..…..transparent cap

●2X Extraction solution 50ml…………………… kapu yabuluu

Kukonzekera kwa Reagents

7.1 Chitsanzo cha uchi

Yankho 1 : 0.2 M Hydrochloric acid solution

Kulemera 41.5ml Concentrated hydrochloric acid, kuchepetsa ndi deionized madzi 500 ml.

Yankho 2: Sambani njira

Sungunulani njira yochapira yokhazikika ndi madzi osungunuka mu chiŵerengero cha 1:19, chomwe chidzagwiritsidwa ntchito kutsuka mbale.yankho kuchepetsedwa akhoza kusungidwa pa 4 ℃ kwa 1 mwezi.

Yankho3: njira yochotsera

Sungunulani 2 × concentrated m'zigawo njira ndi deionized madzi mu voliyumu mlingo wa 1: 1 (kapena kutengera chofunika), amene adzagwiritsidwa ntchito m'zigawo zitsanzo.Njira yothetsera izi imatha kusungidwa kwa mwezi umodzi pa 4 ℃.

Zokonzekera Zitsanzo

8.1 Chidziwitso ndi kusamala kwa ogwiritsa ntchito musanagwire ntchito

(a) Chonde gwiritsani ntchito malangizo amodzi poyesa, ndikusintha malangizowo mukamamwa reagent yosiyana.

(b) Onetsetsani kuti zida zonse zoyesera ndi zoyera, apo ayi zidzakhudza zotsatira zake.

8.2Chitsanzo cha uchi

-----Yezani 2g ± 0.05g uchi wachitsanzo mu chubu cha 50ml polystyrene centrifuge,

-----Onjezani 2ml 0.2 M Hydrochloric acid solution (Yankho 1), vortex kuti musakanize kwathunthu, kenaka yikani 8ml methylene chloride, gwedezani ndi shaker kwa 5min kuti musungunuke kwathunthu;

-----Centrifuge kwa mphindi 10, osachepera 3000g kutentha kwa firiji (20-25 ℃);

-----Chotsani gawo lapamwamba kwambiri, tengani 2 ml ya gawo lapansi la organic solution ku chubu lagalasi la 10 ml. ikani gawo lapansi pansi pa madzi osamba a Nitrogen (50-60 ℃)

-----Onjezani 1 ml n-hexane,vortex kwa 30s, kenaka onjezerani 1ml yothetsera m'zigawo (yankho 3), vortex kachiwiri kwa 1min.Centrifuge kwa 5min, osachepera 3000g kutentha (20-25 ℃);

-----Chotsani gawo lapamwamba, tengani 50ml kuti muyese;

9. Njira yoyesera

9.1 Zindikirani musanayese

9.1.1 Onetsetsani kuti ma reagents ndi ma microwell onse ali pamalo otentha (20-25 ℃).

9.1.2 Bwezerani zotsalira zonse ku 2-8 ℃ mukangogwiritsa ntchito.

9.1.3 Kutsuka ma microwell molondola ndi gawo lofunikira pakuyesa;ndicho chofunikira kwambiri pakubwerezabwereza kwa kusanthula kwa ELISA.

9.1.4 Pewani kuyatsa ndikuphimba tinthu tating'onoting'ono tomwe timayatsa.

9.2 Njira Zoyesera

9.2.1 Tulutsani ma reagents onse kutentha (20-25 ℃) kwa mphindi zopitilira 30, homogenize 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:Nambala malo aliwonse a microwell ndi miyezo yonse ndi zitsanzo ziyenera kuyendetsedwa mobwerezabwereza.Lembani miyeso ndi malo a zitsanzo.

9.2.5Onjezani yankho lokhazikika/chitsanzo:Onjezani 50 µl ya yankho lokhazikika kapena zitsanzo zokonzedwa kuzitsime zofananira.Onjezani 50µl antibody solution.Sakanizani mofatsa pogwedeza mbaleyo pamanja ndikuumirira kwa 30min pa 25 ℃ ndi chophimba.

9.2.6Sambani:Chotsani chivundikiro pang'onopang'ono ndikuyeretsa madziwo m'zitsime ndikutsuka ma microwells ndi 250µl wothira wosambitsa wothira (njira 2) pakadutsa mphindi 10 kwa 4-5.Yankhani madzi otsalawo ndi pepala loyamwa (mpweya wotsalawo ukhoza kuthetsedwa ndi nsonga yosagwiritsidwa ntchito).

9.2.8.Kuphatikiza kwa enzyme:Onjezani enzyme conjugate solution 100ml pachitsime chilichonse, Sakanizani pang'onopang'ono pogwedeza mbale pamanja ndikuumirira kwa 30min pa 25 ℃ ndi chophimba.Bwerezaninso sitepe yochapa kachiwiri.

9.2.8Mitundu:Onjezani 50µl yankho A ndi 50µl yankho B pachitsime chilichonse.Sakanizani pang'onopang'ono pogwedeza mbale pamanja ndikuumirira kwa mphindi 15 pa 25 ℃ ndi chophimba (onani 12.8).

9.2.9Yesani:Onjezani 50µl yankho loyimitsa pachitsime chilichonse.Sakanizani pang'onopang'ono pogwedeza mbale pamanja ndi kuyeza kuyamwa kwa 450nm motsutsana ndi mpweya wopanda kanthu (Ndiyezo muyeso ndi wavelength wapawiri wa 450/630nm. Werengani zotsatira mkati mwa 5min mutawonjezera njira yoyimitsa. ) (Tikhozanso kuyeza ndi kuona. popanda kuyimitsa njira mwachidule cha ELIASA chida)

Zotsatira

10.1 Peresenti ya absorbance

Ziwerengero zamtengo wapatali zomwe zimatengedwa pamiyeso ndi zitsanzo zimagawidwa ndi mtengo wamtengo wapatali wamtundu woyamba (zero standard) ndikuchulukitsa ndi 100%.Muyezo wa zero umapangidwa kukhala wofanana ndi 100% ndipo milingo ya kuyamwa imatchulidwa mwamaperesenti.

Absorbance (%) = B/B0 × 100%

B --absorbance muyezo (kapena chitsanzo)

B0 --absorbance zero muyezo

10.2 Njira Yokhazikika

Kuti mujambule mapindikidwe wamba: Tengani kuchuluka kwa milingo monga y-axis, semi logarithmic of the concentration of the flumequine standards solution (ppb) monga x-axis.

--- Theflumequinekuchuluka kwa zitsanzo zilizonse (ppb), zomwe zitha kuwerengedwa kuchokera pamapindikira owerengera, zimachulukitsidwa ndi kuphatikizika kofananira kwachitsanzo chilichonse chotsatiridwa, ndipo kuchuluka kwenikweni kwa zitsanzo kumapezedwa.

Pofuna kuchepetsa deta ya zida za ELISA, mapulogalamu apadera apangidwa, omwe angaperekedwe popempha.

11. Kumverera, kulondola ndi kulondola

Kumverera kwa Mayeso:0.3ppb

Uchi Chitsanzo cha dilution factor: 2

Malire ozindikira

Zitsanzo za uchi----------------------------------------------- -1ppb

Kulondola

Zitsanzo za uchi -------------------------------------------- 90±20 %

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 kubwerezabwereza kosatheka ndikugwiritsanso ntchito sitepe yotsatira mukangogwira chosungira ma microwells.

12.3.Homogenize aliyense reagent musanagwiritse ntchito.

12.4.Sungani khungu lanu kutali ndi njira yoyimitsa chifukwa ndi 2M H₂SO₄yankho.

12.5 Musagwiritse ntchito zida zakale.Osasinthanitsa ma reagents amagulu osiyanasiyana, chifukwa amatsitsa chidwi.

12.6 Malo osungira:

Sungani zida za ELISA pa 2-8 ℃, osazizira.Tsekani mbale zopumira zopumira Pewani kuwala kwadzuwa nthawi zonse mu incubation.Kuphimba mbale microtiter tikulimbikitsidwa.

12.7 Zizindikiro zakuwonongeka kwa ma reagents:

Njira yapansi panthaka iyenera kusiyidwa ngati isintha mitundu.

Ma reagents atha kukhala oyipa ngati kuchuluka kwa kuyamwa (450/630nm) kwa muyezo wa ziro ndikochepera 0.5 (A450nm＜0.5).

12.8 Maonekedwe amtundu amafunikira 15min mutatha kuwonjezera Solution A ndi Solution B. Ndipo mukhoza kutalikitsa nthawi ya incubation kuyambira 20min kupita ku zambiri ngati mtunduwo ndi wopepuka kwambiri kuti udziwike.Osapitirira 25min, M'malo mwake, chepetsani nthawi yoyamwitsa bwino.

12.9 The mulingo woyenera kwambiri kutentha ndi 25 ℃.Kutentha kwakukulu kapena kutsika kumayambitsa kusintha kwa chidwi ndi kuyamwa.

13. Kusungirako

Malo osungira: 2-8 ℃.

Nthawi yosungira: miyezi 12.