ngwaahịa

Ụkpụrụ ule

Ngwa a dabere na teknụzụ ELISA na-asọmpi na-apụtaghị ìhè.A na-ekpuchi olulu mmiri microtiter na antigen njikọ.Flumequineihe fọdụrụ n'ime ihe nlele ahụ na-asọrịta mpi na antigen mkpuchi na efere microtiter maka mgbochi.Mgbe mgbakwunye nke enzyme akpọrọ anti-antibody, a na-eji mkpụrụ TMB gosi agba.Mwepu nke ihe nlele ahụ na-enwe mmetụta na-adịghị mma na tetracycline bi na ya, mgbe atụnyere ya na Standard Curve, mụbaa site na dilution multiple, Flumequine residue quantity na sample nwere ike gbakọọ.

Ngwa

Enwere ike iji ngwa a na nyocha ọnụọgụ na nyocha nke flumequine fọdụrụ na mmanụ aṅụ.

Mmeghachi omume

Flumequine …………………………………………………………………………………………………………………

Ihe achọrọ

Akụrụngwa

┅┅ Igwe nleba anya igwe microtiter (450nm/630nm)

┅┅Homogenizer ma ọ bụ afo

┅┅Shaker

┅┅ Vortex igwekota

┅┅Centrifuge

┅┅ Ntụle nyocha (inductance: 0.01g)

┅┅ Pipette emechara: 15ml

┅┅ bọlbụ pipette rubber

┅┅ Polystyrene Centrifuge tube: 15ml, 50ml

┅┅ tube ule enyo: 10ml

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

250 ml - multipipette

Reagents

┅┅n-hexane (AR)

┅┅Methylene chloride (AR)

┅┅ acetonitrile (AR)

┅┅ Mmiri a na-agbapụta

---Hydrochloric acid (AR) gbadoro ụkwụ

Ngwa ngwa

● Microtiter efere nwere olulu mmiri 96 nke ejiri antigen kpuchie

● Standard ngwọta (6 karama × 1ml / karama)

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

● Nnukwu nchịkwa ụkpụrụ nchịkwa: (1ml / karama)

…………………………………………………………………………………100pb

● Enzyme conjugate 12ml………………………………. okpu uhie

● Antibody ngwọta 7ml ………………………………………….. okpu akwụkwọ ndụ akwụkwọ ndụ

● Ngwọta A 7ml………………………………………………………….. okpu ọcha

● Ngwọta B 7ml ……………………………………………………. okpu uhie

● Kwụsị ngwọta 7ml …………………………………………………………. okpu edo edo

● 20XConcentrated saa ngwọta 40ml

……………………………………………………………………

●2X ngwọta mmịpụta 50ml………………………………. okpu na-acha anụnụ anụnụ

Nkwadebe Reagent

7.1 Honey sample

Ngwọta 1: 0.2M Hydrochloric acid ngwọta

Ibu 41.5ml Concentrated hydrochloric acid, gbanye ya na mmiri deionized ruo 500 ml.

Ngwọta 2: Ngwọta saa

Gwakọta ihe ngwọta na-edozi ahụ na mmiri deionized na nha nha nke 1:19, nke a ga-eji saa efere.Enwere ike ịchekwa ngwọta diluted na 4 ℃ maka ọnwa 1.

Ngwọta3: ngwọta mmịpụta

Ghichaa ngwọta mmịpụta nke 2 × na mmiri deionized na oke olu nke 1: 1 (ma ọ bụ dabere na ihe achọrọ), nke a ga-eji maka mmepụta ihe atụ.Nke a diluted ngwọta nwere ike chekwa maka 1 ọnwa na 4 ℃.

Nkwadebe ihe atụ

8.1 Ọkwa na akpachapụ anya maka ndị ọrụ tupu arụ ọrụ

(a) Biko jiri ndụmọdụ na-apụ apụ na usoro nnwale, ma gbanwee ndụmọdụ mgbe ị na-etinye reagent dị iche iche.

(b) Jide n'aka na ngwa nnwale niile dị ọcha, ma ọ bụghị ya, ọ ga-emetụta nsonaazụ nyocha.

8.2Ihe atụ mmanụ aṅụ

--atụ 2g± 0.05g mmanụ aṅụ n'ime tube 50ml polystyrene centrifuge,

---Tinye 2ml 0.2M Hydrochloric acid solution (Ngwọta 1), vortex ka ị gwakọta ya kpamkpam, wee tinye 8ml methylene chloride, maa jijiji na shaker maka 5min ka ọ gbazee kpamkpam;

--Centrifuge maka 10 min, opekata mpe 3000g na ụlọ okpomọkụ (20-25 ℃);

- - - -Wepụ ihe na-enweghị isi, were 2 ml nke mkpụrụ osisi organic na tube iko iko 10 ml.

---Tinye 1 ml n-hexane, vortex maka 30s, wee gbakwunye ngwọta mmịpụta 1ml (ihe ngwọta 3), vortex ọzọ maka 1min.Centrifuge maka 5min, ọ dịkarịa ala 3000g na ụlọ okpomọkụ (20-25 ℃);

--Wepu usoro nke elu, were 50ml maka nyocha;

9. Usoro nyocha

9.1 Rịba ama tupu nyocha

9.1.1 Jide n'aka na ihe niile reagents na microwells niile dị na ụlọ okpomọkụ (20-25 ℃).

9.1.2 Weghachite ihe niile fọdụrụ reagents na 2-8 ℃ ozugbo ejiri ya.

9.1.3 Ịsacha microwells nke ọma bụ nzọụkwụ dị mkpa na usoro nyocha;ọ bụ ihe dị mkpa maka nyochaghachi ELISA ugboro ugboro.

9.1.4 Zere ọkụ ma kpuchie microwells n'oge incubation.

9.2 Usoro nyocha

9.2.1 Wepụ ihe niile reagents na ụlọ okpomọkụ (20-25 ℃) maka ihe karịrị 30min, homogenize tupu ojiji.

9.2.2 Nweta microwells achọrọ wee weghachi ndị ọzọ n'ime akpa mkpọchi zip na 2-8 ℃ ozugbo.

9.2.3 Ngwọta ịsacha a gwakọtara ga-emerịrị ka ọ nọrọ n'ime ụlọ okpomọkụ tupu ejiri ya.

9.2.4Nọmba:Kpọọ nọmba ọnọdụ microwell ọ bụla yana ụkpụrụ na nlele niile ga-agba ọsọ n'ụdị oyiri.Dekọọ ụkpụrụ na ọnọdụ nlele.

9.2.5Tinye ihe ngwọta/ụkpụrụ ọkọlọtọ:Tinye 50 µl nke ngwọta ọkọlọtọ ma ọ bụ ihe nlele akwadoro na olulu mmiri kwekọrọ.Tinye 50µl ngwọta mgbochi mmadụ.Gwakọta nke ọma site na iji aka na-amagharị efere ahụ ma tinye ya maka 30min na 25 ℃ na mkpuchi.

9.2.6saa:Wepu ihe mkpuchi ahụ nke ọma ma kpochaa mmiri mmiri ahụ n'olulu mmiri ma kpochaa microwells na ngwọta 250µl diluted (ngwọta 2) na etiti nkeji 10 maka oge 4-5.Were akwụkwọ na-amịkọrọ mmiri na-amịpụta mmiri fọdụrụnụ (enwere ike iwepụ afụ ikuku ndị ọzọ site n'ọnụ ọnụ na-ejighị ya).

9.2.8.Enzyme conjugate:Tinye ihe ngwọta conjugate enzyme 100ml na olulu mmiri ọ bụla, gwakọta nwayọọ nwayọọ site na iji aka na-amagharị efere ahụ ma tinye maka 30min na 25 ℃ na mkpuchi.Tinyegharịa nzọụkwụ ịsa ahụ ọzọ.

9.2.8Agba:Tinye 50µl ngwọta A na 50µl ngwọta B na nke ọ bụla.Gwakọta nke ọma site na iji aka na-amagharị efere ahụ ma tinye ya maka nkeji 15 na 25 ℃ na mkpuchi (lee 12.8).

9.2.9tụọ:Tinye 50µl ngwọta nkwụsị n'olulu mmiri ọ bụla.Gwakọta nke ọma site n'iji aka na-amagharị efere ahụ ma tụọ nnabata ahụ na 450nm megide oghere ikuku (A na-atụ aro ya na ọnụọgụ abụọ-wavelength nke 450 / 630nm. Gụọ nsonaazụ n'ime 5min mgbe mgbakwunye nke nkwụsị nkwụsị. ) (Anyị nwekwara ike ịlele site na anya). na-enweghị nkwụsị ngwọta na nkenke nke ELIASA ngwá ọrụ)

Nsonaazụ

10.1 pasent nnabata

The pụtara ụkpụrụ nke absorbance ụkpụrụ enwetara maka ụkpụrụ na samples na-ekewa site absorbance uru nke mbụ ọkọlọtọ (efu ọkọlọtọ) na mụbaa site 100%.A na-eme ọkọlọtọ efu ka ọ bụrụ 100% yana ụkpụrụ nnabata na-ehota na pasentị.

Mmanya (%) = B/B0 × 100%

B ——ụkpụrụ nnabata (ma ọ bụ sample)

B0 ——ụkpụrụ efu efu

10.2 Standard Curve

Iji see usoro ọkọlọtọ: Were uru nnabata nke ụkpụrụ dị ka y-axis, ọkara logarithmic nke ntinye uche nke ụkpụrụ flumequine (ppb) dị ka x-axis.

--- Nkeflumequinentinye uche nke ihe nlele ọ bụla (ppb), nke enwere ike ịgụ site na nhazi nhazi, na-abawanye site na ntinye dilution kwekọrọ na nke ọ bụla n'ime ihe nlele ọ bụla na-esote, a na-enwetakwa ntinye uche n'ezie.

Maka mbelata data nke ngwa ELISA, emepụtara sọftụwia pụrụ iche, nke enwere ike ịnye na arịrịọ.

11. Mmetụta, izi ezi na nkenke

Nwalee mmetụta:0,3pb

Ihe nhụta mmanụ aṅụ sample dilution ihe: 2

Oke nchọpụta

Ihe atụ mmanụ aṅụ --------------------------------- -1 pb

izi ezi

Ihe atụ mmanụ aṅụ -------------------------------------------------- 90±20 %

Nkenkenke

Ọnụego mgbanwe nke ngwa ELISA erughị 10%.

12. Mara

12.1 The pụtara ụkpụrụ nke absorbance ụkpụrụ enwetara maka ụkpụrụ na samples ga-ebelata ma ọ bụrụ na reagents na samples e regulated ka ụlọ okpomọkụ (20-25 ℃).

12.2 Ekwela ka microwells kpoo n'etiti usoro iji zere ịmegharị ugboro ugboro na-eme nke ọma ma rụọ ọrụ na-esote ozugbo ị kpatụ ihe njide microwells.

12.3.Homogenize ọ bụla reagent tupu iji.

12.4.Debe akpụkpọ gị pụọ na ngwọta nkwụsị n'ihi na ọ bụ 2M H₂SO₄ngwọta.

12.5 Ejila ngwa ndị ahụ emechiela.Agbanwela reagents nke batches dị iche iche, n'ihi na ọ ga-adaba uche.

12.6 Ọnọdụ nchekwa:

Debe ngwa ELISA na 2-8 ℃, ajụla oyi.Mechie efere microwell izu ike Zere ìhè anyanwụ kwụ ọtọ n'oge nnabata niile.A na-atụ aro ikpuchi efere microtiter.

12.7 Ngosipụta maka reagents na-aga nke ọma:

A ga-ahapụrịrị ngwọta mkpụrụ osisi ma ọ bụrụ na ọ na-atụgharị agba.

Ndị reagents nwere ike gbanwee ihe ọjọọ ma ọ bụrụ na uru nnabata (450/630nm) nke ọkọlọtọ efu bụ ihe na-erughị 0.5 (A450nm ~ 0.5).

12.8 Mmeghachi omume agba chọrọ 15min mgbe agbakwunyere Ngwọta A na Ngwọta B. Ma ị nwere ike ịgbatị oge ntinye oge sitere na 20min ruo karịa ma ọ bụrụ na agba dị oke ọkụ ka a chọpụta ya.Agafela 25min, Kama nke ahụ, belata oge nnabata nke ọma.

12.9 ezigbo mmeghachi omume okpomọkụ bụ 25 ℃.Okpomọkụ dị elu ma ọ bụ dị ala ga-eduga na mgbanwe nke uche na ụkpụrụ nnabata.

13. Nchekwa

Ọnọdụ nchekwa: 2-8 ℃.

Oge nchekwa: ọnwa iri na abụọ.