huahana

Kumu Hoao

Hoʻokumu ʻia kēia pahu i ka ʻenehana ELISA hoʻokūkū kūʻokoʻa.Hoʻopili ʻia nā pūnāwai microtiter me nā antigen hoʻohui.FlumequineʻO ke koena o ka hāpana e hoʻokūkū me ka antigen i uhi ʻia ma ka pā microtiter no ka antibody.Ma hope o ka hoʻohui ʻana o ka enzyme i kapa ʻia he anti-antibody, hoʻohana ʻia ka substrate TMB e hōʻike i ke kala.Absorbance o ka hāpana pili maikaʻi i ka tetracycline noho i loko, ma hope o ka hoohalike ana me ka Standard Curve, hoonuiia e ka dilution lehulehu, Flumequine koena nui i loko o ka hāpana hiki ke helu.

Nā noi

Hiki ke hoʻohana ʻia kēia pahu i ka quantitative a qualitative analysis of flumequine koena i loko o ka meli.

Nā hoʻololi keʻa

Flumequine ……………………………………………………… 100%

Pono Pono

Mea lako

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

┅┅Homogenizer a i ʻole ʻōpū

┅┅mea lulu

┅┅Vortex hui pū

┅┅Centrifuge

┅┅Analytical kaulike (inductance: 0.01g)

┅┅Pipette puka puka: 15ml

┅┅Upu paipu Rubber

┅┅Polystyrene Centrifuge paipu: 15ml, 50ml

┅┅Papa hōʻike aniani:10ml

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

250ml -multipipette

Nā mea hoʻopaʻa

┅┅n-hexane(AR)

┅┅Methylene chloride(AR)

┅┅Acetonitrile(AR)

┅┅Wai hoʻoheheʻe ʻia

-----ʻakika hydrochloric paʻa (AR)

Nā ʻāpana pahu

● ʻO ka pā Microtiter me nā pūnāwai 96 i uhi ʻia me ka antigen

● Nā hāʻina maʻamau (6 ʻōmole × 1ml / ʻōmole)

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

● Kiʻekiʻe manaʻo mana maʻamau: (1ml /'ōmole)

…………………………………………………………………100ppb

● Enzyme conjugate 12ml………………………………

● ʻO ka hoʻonā antibody 7ml …………..………..……..pale ʻōmaʻomaʻo

● Hoʻonā A 7ml……..…………………..……..pale keʻokeʻo

● Hoʻonā B 7ml …………………………………………………… ʻulaʻula

● Hoʻopau i ka solution 7ml ………………………………………

● 20XConcentrated holoi holoi 40ml

……………………………………………...palena alohilohi

●2X ʻO ka hoʻoheheʻe ʻana 50ml……………………… pāpale polū

Hoʻomākaukau Reagents

7.1 Laʻana meli

Haʻina 1 : 0.2 M Hūnā waika

Kaumaha 41.5ml Aakika hydrochloric paʻa, dilute me ka wai deionized i 500 ml.

Pane 2: Holoi holoi

E hoʻoheheʻe i ka hopena holoi holoi me ka wai deionized i ka ratio volume o 1:19, kahi e hoʻohana ai no ka holoi ʻana i nā papa.Hiki ke mālama ʻia ka hopena diluted ma 4 ℃ no 1 mahina.

Hoʻoholo3: hoʻonā unuhi

E hoʻoheheʻe i ka 2×concentrated extraction solution me ka wai deionized i ka nui ration o 1:1 (a i ʻole e hilinaʻi ʻia i ka koi), e hoʻohana ʻia no ka lawe ʻana i ka laʻana.Hiki ke mālama ʻia kēia hopena i hoʻoheheʻe ʻia no 1 mahina ma 4 ℃.

Hoʻomākaukau Laʻana

8.1 Hoʻolaha a me ka mālama ʻana i nā mea hoʻohana ma mua o ka hana

(a) E ʻoluʻolu e hoʻohana i nā ʻōlelo aʻoaʻo hoʻokahi i ke kaʻina hana o ka hoʻokolohua, a hoʻololi i nā ʻōlelo aʻoaʻo i ka wā e komo ai i nā reagent like ʻole.

(b) E hōʻoia i ka maʻemaʻe o nā mea hoʻokolohua a pau, i ʻole e hopena ia i ka hopena hoʻokolohua.

8.2Laʻana meli

----- Kauona 2g ± 0.05g honey sample i loko o ka 50ml polystyrene centrifuge paipu,

-----E hoʻohui i 2ml 0.2 M Hydrochloric acid solution (Solution 1), vortex e hui pū me ia, a laila e hoʻohui i ka 8ml methylene chloride, e lulu me ka lulu no 5min e hemo loa;

-----Centrifuge no 10 min, ma kahi o 3000g ma ka lumi wela (20-25 ℃);

----- Wehe i ka mahele supernatant, e lawe i 2 ml o ka substrate organic solution i ka 10 ml aniani paipu.

-----Hoʻohui i 1 ml n-hexane, vortex no 30s, a laila hoʻohui i 1ml hoʻonā hoʻoneʻe (hoʻonā 3), vortex hou no 1min.Centrifuge no 5min, ma kahi o 3000g ma ka lumi wela (20-25 ℃);

----- Wehe i ka supernatant phase, e lawe i 50ml no ka assay;

9. Kaʻina hoʻokolohua

9.1 Hoʻolaha ma mua o ka hoʻāʻo ʻana

9.1.1 E hoʻopaʻa pono i nā reagents a me nā microwell a pau ma ka lumi wela (20-25 ℃).

9.1.2 E hoʻihoʻi i nā mea hoʻoponopono i koe i 2-8 ℃ ma hope koke o ka hoʻohana ʻana.

9.1.3 ʻO ka holoi pono ʻana i nā microwells kahi hana koʻikoʻi i ke kaʻina hana hoʻāʻo;ʻo ia ke kumu koʻikoʻi i ka hana hou ʻana o ka ʻike ELISA.

9.1.4 E pale i ka malamalama a e uhi i na microwells i ka wa e hoohua ai.

9.2 Nā ʻanuʻu hōʻike

9.2.1 E lawe i nā mea hou a pau i waho ma ka lumi wela (20-25 ℃) no ka ʻoi aku ma mua o 30min, homogenize ma mua o ka hoʻohana ʻana.

9.2.2 E kiʻi i nā microwells pono a hoʻihoʻi i ke koena i loko o ka ʻeke paʻa zip ma 2-8 ℃ koke.

9.2.3 Pono e hoʻomaʻamaʻa hou ʻia ka mea holoi holoi i hoʻoheheʻe ʻia ma ka lumi wela ma mua o ka hoʻohana ʻana.

9.2.4Helu:E helu i kēlā me kēia kūlana microwell a me nā maʻamau a me nā laʻana e holo pālua.E hoʻopaʻa i nā kūlana maʻamau a me nā laʻana.

9.2.5Hoʻohui i ka hāʻina maʻamau/laʻana:E hoʻohui i 50 µl o ka hopena maʻamau a i ʻole ka laʻana i hoʻomākaukau ʻia i nā pūnāwai pili.E hoʻohui i 50µl i ka hopena antibody.E hoʻokuʻi mālie me ka lulu ʻana i ka pā me ka lima a hoʻomoʻa no 30 mau minuke ma 25 ℃ me ka uhi.

9.2.6Holoi:Wehe mālie i ka uhi a hoʻomaʻemaʻe i ka wai mai loko mai o nā pūnāwai a holoi i nā microwells me 250µl diluted wash solution (solution 2) ma kahi o 10s no 4-5 mau manawa.E hoʻopau i ke koena wai me ka pepa hoʻoheheʻe (hiki ke hoʻopau ʻia ke koena o ka ea me ka ʻaoʻao i hoʻohana ʻole ʻia).

9.2.8.Enzyme conjugate:E hoʻohui i ka enzyme conjugate solution 100ml i kēlā me kēia pūnāwai, E hui mālie me ka lulu ʻana i ka pā me ka lima a hoʻomoʻa no 30min ma 25 ℃ me ka uhi.E hana hou i ka hana holoi.

9.2.8kala kala:E hoʻohui i 50µl solution A me 50µl solution B i kēlā me kēia pūnāwai.E hoʻokuʻi mālie me ka lulu ʻana i ka pā me ka lima a hoʻomoʻa no 15 min ma 25 ℃ me ka uhi (e nānā i ka 12.8).

9.2.9Ana:E hoʻohui i 50µl i ka hopena hoʻomaha i kēlā me kēia pūnāwai.E hoʻokuʻi mālie ma ka lulu lima ʻana i ka pā a e ana i ka absorbance ma 450nm e kūʻē i kahi ea blank (Ua manaʻo ʻia e ana ʻia me ka lōʻihi-lua o 450/630nm. E heluhelu i ka hopena i loko o 5min ma hope o ka hoʻohui ʻana i ka hopena hoʻomaha. ) (Hiki iā mākou ke ana ma ka ʻike. me ka hoʻopau ʻole ʻana i ka hopena i ka pōkole o ka mea hana ELIASA)

Nā hualoaʻa

10.1 Pākēneka o ke komo ʻana

Hoʻokaʻawale ʻia nā kumukūʻai kumu o nā waiwai absorbance i loaʻa no nā maʻamau a me nā hōʻailona e ka waiwai absorbance o ka maʻamau mua (zero standard) a hoʻonui ʻia e 100%.No laila, ua like ka zero standard me 100% a ua helu ʻia nā helu absorbance ma nā pākēneka.

ʻAi ʻana (%) = B/B0 ×100%

B ——absorbance maʻamau (a i ʻole ka laʻana)

B0 ——absorbance zero standard

10.2 Pipi maʻamau

No ke kaha ki'i ma'amau: E lawe i ka waiwai ho'omohu o nā ma'amau ma ke 'ano he axis-y, semi logarithmic o ka ne'e 'ana o ka solution standards flumequine (ppb) ma ke 'ano he-x.

--- KaflumequineHoʻonui ʻia ka neʻe ʻana o kēlā me kēia hāpana (ppb), hiki ke heluhelu ʻia mai ka ʻōkuhi calibration, e ka helu hoʻoheheʻe like ʻana o kēlā me kēia hāpana i ukali ʻia, a loaʻa ka ʻike maoli o ka hāpana.

No ka hōʻemi ʻikepili o nā kits ELISA, ua hoʻomohala ʻia nā polokalamu kūikawā, hiki ke hāʻawi ʻia ma ke noi.

11. Ka noʻonoʻo, ka pololei a me ka pololei

Hoao Hoao：0.3ppb

Honey laʻana hoʻoheheʻe kumu: 2

palena ʻike

Honey sample------------------------------------------------ -1ppb

Ka pololei

Laʻana meli ------------------------------------------------- 90±20 %

pololei

ʻOi aku ka liʻiliʻi o ka helu hoʻololi o ka pahu ELISA ma mua o 10%.

12. Hoolaha

12.1 E hoʻemi ʻia nā kumu waiwai o nā waiwai absorbance i loaʻa no nā maʻamau a me nā laʻana inā ʻaʻole i hoʻoponopono ʻia nā reagents a me nā laʻana i ka lumi wela (20-25 ℃).

12.2 Mai ʻae i nā microwells e maloʻo ma waena o nā ʻanuʻu e pale i ka hana hou ʻole ʻana a hana i ka ʻanuʻu aʻe ma hope koke o ka paʻi ʻana i ka pahu microwells.

12.3.Homogenize kēlā me kēia reagent ma mua o ka hoʻohana ʻana.

12.4.E mālama i kou ʻili mai ka hopena hoʻomaha no ka mea ʻo ia ka 2M H₂SO₄hoʻonā.

12.5 Mai hoʻohana i nā kit i ka wā kahiko.Mai hoʻololi i nā reagents o nā ʻāpana like ʻole, no ka mea, e hāʻule ana ka naʻau.

12.6 Kūlana mālama:

E mālama i nā pahu ELISA ma 2-8 ℃, mai maloʻo.Hoʻopaʻa sila i nā papa microwell hoʻomaha.Paipai ʻia ka uhi ʻana i nā papa microtiter.

12.7 Nā hōʻailona no nā reagents e hele hewa ana:

Pono e haʻalele ʻia ka hoʻonā substrate inā huli ia i nā kala.

He ʻino paha nā reagents inā ʻoi aku ka liʻiliʻi o ke kumu kūʻai (450/630nm) o ka maʻamau zero ma mua o 0.5 (A450nm <0.5).

12.8 Pono ka hopena kala i ka 15min ma hope o ka hoʻohui ʻana i ka Solution A a me ka Solution B. A hiki iā ʻoe ke hoʻolōʻihi i ka manawa incubation mai 20min a ʻoi aku inā he māmā ke kala e hoʻoholo ai.Mai ʻoi aku ma mua o 25min, akā, e hoʻopōkole pono i ka manawa incubation.

12.9 ʻO ka mahana hopena maikaʻi loa he 25 ℃.ʻO ke kiʻekiʻe a i ʻole ka haʻahaʻa haʻahaʻa e alakaʻi i nā loli o ka naʻau a me nā waiwai absorbance.

13. Waihona

Kūlana mālama: 2-8 ℃.

Ka manawa mālama: 12 mahina.