Asọmpi Enzyme Immunoassay Kit maka Quantitative Analysis of Tylosin
Ngwa Enzyme Immunoassay nke asọmpi maka
Quantitative Analysis nkeTylosin
1. ndabere
Tylosinbụ ọgwụ nje macrolide, nke a na-ejikarị dị ka antibacterial na anti-mycoplasma.E guzobela MRL siri ike ebe ọ bụ na ọgwụ a nwere ike ibute mmetụta dị njọ na ụfọdụ otu.
Ngwa a bụ ngwaahịa ọhụrụ dabere na teknụzụ ELISA, nke dị ngwa, dị mfe, ziri ezi ma nwee mmetụta ma e jiri ya tụnyere nyocha ngwa ngwa na-achọ naanị awa 1.5 n'otu ọrụ, ọ nwere ike belata njehie ọrụ yana ike ọrụ.
2. Ụkpụrụ ule
Ngwa a dabere na teknụzụ ELISA na-asọmpi na-apụtaghị ìhè.A na-ekpuchi olulu mmiri microtiter na antigen njikọ.Ihe fọdụrụ Tylosin n'ime ihe nlele ahụ na-asọ 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 tylosin bi n'ime ya, mgbe atụnyere ya na Standard Curve, mụbaa site na ihe dilution, enwere ike gbakọọ ọnụọgụ nke tylosin n'ime ihe nlele ahụ.
3. Ngwa
Enwere ike iji ngwa a mee ihe na nyocha ọnụọgụ na qualitative nke tylosin residue na anụ anụ (ọkụkọ, anụ ezi, ọbọgwụ) na mmiri ara ehi, mmanụ aṅụ, akwa, wdg.
4. Mmeghachi omume
Tylosin………………………………………………………………………………………………………………………….
Tilmicosin………………………………………………………………………………………………………
5. Ihe achọrọ
5.1 Akụrụngwa:
---- Mpekere spectrophotometer nke Microtiter (450nm/630nm)
---- Rotary evaporator ma ọ bụ akụrụngwa ihicha nitrogen
---- homogenizer
----Shaker
----Centrifuge
---- Ntụle nyocha (inductance: 0.01g)
---- pipette emechara: 10ml
---- bọlbụ pipette
---- Mpekere olu: 10ml
----Polystyrene centrifuge tube: 50ml
---- Micropipettes: 20-200ml, 100-1000ml
250 ml - multipipette
5.2 Reagent:
----Sodium hydroxide (NaOH, AR)
---- sodium bicarbonate (NaHCO3,AR)
---- sodium carbonate (NaCO3, AR)
----Trichloroacetic acid (AR)
---- Acetonitrile (AR)
----Ethyl acetate (AR)
┅┅n-Hexane (AR)
----Mmiri a na-agbapụta
6. Ngwa ngwa
l Microtiter efere nwere olulu mmiri 96 nke ejiri antigen kpuchie ya
l Standard ngwọta (5 karama, 1ml / karama)
0ppb, 0.5ppb, 1.5ppb, 4.5ppb, 13.5ppb
l nchịkwa ọkọlọtọ: (1ml / karama)1ppm
l Enzyme conjugate 1ml………………………………………. okpu uhie
l Antibody ngwọta 7ml………………………………. okpu akwụkwọ ndụ akwụkwọ ndụ
l Ngwọta A 7ml…………………………………………………………. okpu ọcha
l Ngwọta B 7ml……………………………………………….. okpu uhie
l Kwụsị ngwọta 7ml………………………………. okpu edo edo
l 20 × lekwasị asacha ngwọta 40ml
………………………………………………………………
l 4 × ndọpụ mmịpụta ngwọta 50ml
……………………………………………………………………………………………….Okpu na-acha anụnụ anụnụ
7. Nkwadebe Reagent:
Ngwọta 1:0.1mol/L NaOH ngwọta
Tua 0.4g NaOH ruo 100ml mmiri deionized wee gwakọta ya kpamkpam.
Ngwọta 2: 1mol/L NaOH ngwọta
Tua 4g NaOH ruo 100ml mmiri deionized wee gwakọta ya kpamkpam.
Ngwọta 3: Carbonate buffer nnu
Ngwọta1: 0.2M PB
Gbanyụọ 51.6g nke Na2HPO4· 12H2O, 8.7g nke NaH2PO4· 2H2O na mmiri deionized na itughari ka 1000ml.
Ngwọta2: ngwọta mmịpụta
Ghichaa ihe ngwọta mmịpụta 2 × na mmiri deionized na oke olu nke 1: 1 (eg 10ml nke 2 × mmịpụta ngwọta + 10ml nke deionized mmiri), nke a ga-eji maka mmịpụta sample,ngwọta a nwere ike ịchekwa na 4 ℃ maka 1 ọnwa.
Ngwọta3: Ngwọta saa
Jiri mmiri deionized gbanye ihe ngwọta 20 × na-etinye uche na ya na oke olu nke 1:19 (eg 5ml nke 20 × asacha ngwọta + 95ml nke deionized mmiri), nke a ga-eji saa efere.Enwere ike ịchekwa ngwọta a na 4 ℃ maka otu ọnwa.
8. Nkwadebe ihe atụ
8.1 Rịba ama na ịkpachara anya 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) Hụ na ngwá ọrụ niile dị ọcha.
(c) Debe ihe nlele anụ ahụ na friji.
(d) Ekwesịrị iji nlele akwadoro mee nyocha ozugbo.
8.2 anụ ahụ anụ (ọkụkọ, anụ ezi, wdg)
----Homogenize sample na homogenizer;
---- Were 2.0 ± 0.05g nke homogenate n'ime 50ml polystyrene centrifuge tube;tinye 2ml nke 0.2M PB (ngwọta1) , maa jijiji ka igbari, wee tinye 8ml nke ethyl acetate ma maa jijiji nke ukwuu maka 3min;
----Centrifuge maka nkewa: 3000g / ambient okpomọkụ / 5min.
---- Nyefee 4ml nke usoro ihe ọkụkụ dị elu n'ime tube iko 10ml, kpoo ya na mmiri ịsa mmiri 50-60 ℃ n'okpuru iyi gas nitrogen;
---- Gbanyụọ ihe akọrọ akọrọ na 1ml nke n-hexane, vortex maka 30s ka ọ gbazee, wee tinye 1ml nke ngwọta mmịpụta (ngwọta2), vortex maka 1min.centrifuge maka nkewa: 3000g / ambient okpomọkụ / 5min
----Wepu oke n-hexane nkeji;Were 50μl nke mkpụrụ osisi aqueous maka nyocha.
Ihe mgbaze: 1
8.2 Mmiri ara ehi
---- Were 100μl nke raw mmiri ara ehi sample, mix na 900μl nke mmịpụta ngwọta (ngwọta2), na mix kpamkpam.
---- Were 50μl nke ngwọta a kwadebere maka nyocha.
Ihe mgbaze: 10
9. Usoro nyocha
9.1 Rịba ama tupu nyocha
9.1.1Jide n'aka na reagents na microwells niile dị na ụlọ okpomọkụ (20-25 ℃).
9.1.2Weghachite reagents niile fọdụrụ na 2-8℃ozugbo ejiri ya.
9.1.3Ịsacha microwells nke ọma bụ nzọụkwụ dị mkpa na usoro nyocha;Ọ bụ ihe dị mkpa maka mmụgharị nke nyocha ELISA.
9.1.4 Amebie ọkụ ma kpuchie microwells n'oge nnabata.
9.2 Usoro nyocha
9.2.1 Wepụ ihe niile reagents na ụlọ okpomọkụ (20-25 ℃) maka ihe karịrị 30min, maa jijiji nwayọọ 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:Agụnyere ọnọdụ microwell ọ bụla yana ụkpụrụ na nlele niile kwesịrị ịme ya na oyiri.Dekọọ ụkpụrụ na ọnọdụ nlele.
9.2.5Add ọkọlọtọ ngwọta/sample na antibody ngwọta: Tinye 50µl nke ọkọlọtọ ngwọta (((kit nyere)) ma ọ bụ ihe atụ akwadoro na olulu mmiri kwekọrọ.Tinye 50µl nke ngwọta antibody (kit nyere).Gwakọta nke ọma site na iji aka na-amagharị efere ahụ ma tinye ya maka 30min na 37 ℃ na mkpuchi.
9.2.6saa: Wepu mkpuchi ahụ nke ọma ma kpochaa mmiri mmiri ahụ n'olulu mmiri ma kpochaa microwells na ngwọta 250µl diluted (250µl).ngwọta3) na etiti oge nke 10s maka 4-5 ugboro.Were akwụkwọ na-amịkọrọ mmiri na-amịpụta mmiri fọdụrụnụ (enwere ike iwepụ afụ ikuku ndị ọzọ site n'ọnụ ọnụ na-ejighị ya).
9.2.7Tinye enzyme conjugate: Tinye 100ml nke enzyme conjugate ngwọta (kit nyere) na olulu mmiri ọ bụla, gwakọta nke ọma ma tinye ya maka 30min na 37 ℃ na mkpuchi.Tinyegharịa nzọụkwụ ịsa ahụ ọzọ.
9.2.8Agba: Tinye 50µl nke ngwọta A (kit nyerena 50µl nke ngwọta B (kit nyere) olulu mmiri nke ọ bụla.Gwakọta nke ọma na incubate maka 15min na 37 ℃ na mkpuchi.
9.2.9tụọ: Tinye 50µl nke ngwọta nkwụsị (kit nyere) olulu mmiri nke ọ bụla.Gwakọta nke ọma ma tụọ nnabata ahụ na 450nm (A na-atụ aro ka ọ tụọ ya na ogologo wavelength nke 450/630nm. Gụọ nsonaazụ n'ime 5min mgbe gbakwunyere ngwọta nkwụsị).
10. Nsonaazụ
10.1 pasent nnabata
A na-ekewa ụkpụrụ nke ụkpụrụ nnabata nke enwetara maka ụkpụrụ na ihe nlele site na uru nnabata nke ọkọlọtọ mbụ (ọkọlọtọ efu) ma mụbaa site na 100%.A na-eme ọkọlọtọ efu ka ọ bụrụ 100% yana ụkpụrụ nnabata na-ehota na pasentị.
B
Mmanya (%) = —— × 100%
B0
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 ụkpụrụ tylosin (ppb) dị ka x-axis.
---- A na-amụba mkpokọta tylosin nke ihe nlele ọ bụla (ppb), nke enwere ike ịgụ site na nhazi nhazi, na-abawanye site na ihe Dilution nke kwekọrọ na nke ọ bụla na-esote, ma nweta ezigbo ntinye nke sample.
Biko mara:
emepụtara sọftụwia pụrụ iche maka nyocha data, nke enwere ike ịnye na arịrịọ.
11. Mmetụta, izi ezi na nkenke
Nwale Mmetụta:1.5pb
Oke nchọpụta:
Anụ anụ …………………………………………………………………………………………………………………………………………………………………………….1.5ppb Mmiri ara ehi………………………………………………………………………………………………………………………………………………………………………………………….15ppb izi ezi:
Anụ anụ …………………………………………………………………………………………………………… 80±15%
Mmiri ara ehi……………………………………………………………………………………………… 80±10%
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 mmeghari na-enweghị ihe ịga nke ọma ma rụọ ọrụ na-esote ozugbo ka ịpịa ihe njide microwells.
12.3 Kwanye reagent nke ọ bụla nwayọ tupu eji ya.
12.4 Debe akpụkpọ gị pụọ na ngwọta nkwụsị n'ihi na ọ bụ 0.5MH2SO4ngwọta.
12.5 Ejila ngwa ndị ahụ emechiela.Agbanwela reagents nke batches dị iche iche, ma ọ bụghị ya, ọ ga-agbadata uche.
12.6 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 kwesịrị ịhapụ ihe ngwọta nke mkpụrụ osisi ma ọ bụrụ na ọ gbanwee agba.The reagents nwere ike gbanwee ọjọọ ma ọ bụrụ na absorbance uru (450/630nm) nke efu ọkọlọtọ bụ ihe na-erughị 0.5 (A450nm <0.5).
12.8 Mmeghachi omume agba chọrọ 15min mgbe mgbakwunye nke ngwọta A na ngwọta B. Ma ị nwere ike ịgbatị oge ntinye oge ruo 20min ma ọ bụ karịa ma ọ bụrụ na agba dị oke ọkụ iji kpebisie ike.Agafela 30min, kama nke ahụ, belata oge nnabata nke ọma.
12.9 ezigbo mmeghachi omume okpomọkụ bụ 37 ℃.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.
