Qalabka Enzyme Immunoassay ee Tartan ee Falanqaynta Tirada ee Tylosin
Qalabka Enzyme Immunoassay ee tartanka loogu talagalay
Falanqaynta tiradaTilosin
1. Asalkii hore
Tilosinwaa antibiyootiga macrolide, kaas oo inta badan loo isticmaalo sida bakteeriyada iyo anti-mycoplasma.MRLs adag ayaa la sameeyay tan iyo markii dawadani ay u horseedi karto saameyn halis ah kooxaha qaarkood.
Qalabkani waa badeecad cusub oo ku salaysan tignoolajiyada ELISA, kaas oo ah mid dhakhso badan, fudud, sax ah oo xasaasi ah marka la barbar dhigo falanqaynta qalabka caadiga ah oo kaliya u baahan 1.5 saacadood hal hawlgal, waxay si weyn u yareeyn kartaa qaladka hawlgalka iyo xoojinta shaqada.
2. Mabda'a Imtixaanka
Xirmadan waxay ku salaysan tahay tignoolajiyada ELISA ee aan tooska ahayn ee tartanka.Ceelasha microtiter-ka waxaa lagu dahaadhay antigen-ka isku-xidha.Hadhaaga Tylosin ee muunada waxay ku tartameysaa antigen-ka lagu dahaadhay saxanka microtiter ee difaaca jirka.Ka dib marka lagu daro insaymka lagu calaamadiyay anti-antibody, substrate TMB ayaa loo isticmaalaa si loo muujiyo midabka.Nuugista muunada waxay si xun ula xiriirtaa tylosinka ku dhex nool, ka dib marka la barbar dhigo Curve Standard, oo lagu dhufto factor dilution, tirada haraaga tylosin ee muunada waa la xisaabin karaa.
3. Codsiyada
Qalabkan waxa loo isticmaali karaa qiyaas ahaan iyo falanqayn tayo leh oo ku saabsan hadhaaga tylosin ee ku jira unugyada xoolaha (digaagga, doofaarka, shinbiraha) iyo caanaha, malabka, ukunta, iwm.
4. Falcelin-celin
Tylosin………………………………………………………………………………..100%
Tilmicosin………………………………………………………………………………………….
5. Agabka loo baahan yahay
5.1 Qalabka:
----Mikrotiter saxan spectrophotometer (450nm/630nm)
---- Qalabka uumi-baxa rotary-ga ama nitrogen-ka lagu qalajiyo
---- homogenizer
----Shaker
----Centrifuge
---- dheelitirnaanta falanqaynta (inductance: 0.01g)
---- pipette qalin jabiyay: 10ml
----guluubka tuubada caagga ah
----Dharka mugga leh: 10ml
---- Tuubooyinka centrifuge polystyrene: 50ml
----Mikrobipettes: 20-200ml, 100-1000ml
250ml-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)
----Biyo dheecan
6. Qaybaha Kit
l saxan Microtiter leh 96 ceel oo lagu dahaadhay antigen
l xalalka caadiga ah (5 dhalo, 1ml/dhalo)
0ppb, 0.5ppb, 1.5ppb, 4.5ppb, 13.5ppb
l Xakamaynta heerka caadiga ah: (1ml/ dhalo)1pm
l Enzyme conjugate 1ml…………………………………………
l Xalka difaaca jirka 7ml………………………………………. Koofi cagaaran
l Xalka A 7ml ………………………………………………. dabool cad
l Xalka B 7ml………………………………………………
l Jooji xal 7ml.………………………………. Koofi jaalaha ah
l 20 × xal dhaqid oo xoog leh 40ml
…………………………………………………
l 4 × fiirsashada fiirsashada 50 ml
…………………………………………………. Koofi buluug ah
7. Diyaarinta Reagents:
Xalka 1:0.1mol/L NaOH xal
Miisaanka 0.4g NaOH ilaa 100ml oo biyo ah oo la miiray oo si buuxda u qas.
Xalka 2: 1mol/L NaOH xal
Miisaam 4g NaOH ilaa 100ml oo biyo ah oo la miiray oo si buuxda u qas.
Xalka 3: cusbada kaarboonka
Xalka1: 0.2M PB
Kala diri 51.6g ee Na2HPO4·12H2O, 8.7g ee NaH2PO4·2H2O leh biyo deionized oo ku mil 1000ml.
Xalka2: Xalka soo saarista
Ku walaaq 2 × xalka saarista xoogga leh oo leh biyo deionized saamiga mugga 1: 1 (Tusaale ahaan 10ml oo ah 2× saarista xal + 10ml oo biyo fuuqbaxay), kaas oo loo isticmaali doono soo saarista muunad,Xalkan waxa lagu kaydin karaa 4℃ 1 bil.
Xalka3: Xalka dhaq
Ku walaaq 20x xal dhaqid oo xoog leh oo leh biyo deionized saamiga mugga 1:19 (Tusaale ahaan 5ml oo ah 20×mawrka lagu dhaqo + 95ml oo biyo ah oo la miiray), kaas oo loo isticmaali doono in lagu dhaqo saxanka.Xalkan waxa lagu kaydin karaa 4℃ 1 bil.
8. Tusaalaha Diyaarinta
8.1 Ogeysiis iyo taxaddar ka hor inta aan la hawlgelin:
(a) Fadlan isticmaal talooyinka hal-mar ah habka tijaabada, oo beddel talooyinka marka nuugo reagen kala duwan.
(b) Hubi in dhammaan qalabku nadiif yihiin.
(c) Sambalka unugga meel qabow ku hay.
(d) Muunad la diyaariyey waa in hal mar loo adeegsadaa baaritaanka.
8.2 Nudaha xoolaha (digaagga, doofaarka, iwm)
----Isku ekaynta muunada leh homogenizer;
---- Qaado 2.0 ± 0.05g of homogenate galay 50ml tuubo centrifuge polystyrene;ku dar 2ml oo ah 0.2M PB (xal1) , rux si aad u milanto, ka dibna ku dar 8ml of ethyl acetate oo si adag u rux 3min;
----Centrifuge ee kala-soocidda: 3000g / heerkulka deegaanka / 5min.
----Ku wareeji 4ml ee wejiga organic supernatant galay tube galaas 10ml, qallaji 50-60℃ biyaha qubeyska hoos socodka gaaska nitrogen;
----Ku mil hadhaaga engegan 1ml ee n-hexane, vortex ilaa 30s si ay u milmaan, ka dibna ku dar 1ml oo ah xalinta soo saarista (xal2), vortex 1mincentrifuge ee kala-soocidda: 3000g / heerkulka deegaanka / 5min
---- Ka saar heerka sare ee n-hexane;Qaado 50 μl ee wejiga aqueous substrate si aad u tijaabiso.
Qodobka milanka: 1
8.2 Caano
---- Qaado 100μl oo muunad caano cayriin ah, ku qas 900μl oo ah xalintaxal2), oo si buuxda u qas.
---- Qaado 50μl ee xalka la diyaariyey si aad u tijaabiso.
Qodobka milanka: 10
9. Habka qiimaynta
9.1 Ogaysii ka hor inta aan qiimayn
9.1.1Hubi in dhammaan reajentiyeyaasha iyo microwells ay dhamaantood ku jiraan heerkulka qolka (20-25 ℃).
9.1.2Ku soo celi dhammaan reaageyaasha inteeda kale 2-8℃isla markiiba ka dib marka la isticmaalo.
9.1.3Si sax ah u dhaqida ceelasha yaryar waa tallaabo muhiim u ah habka qiimaynta;waa qodobka muhiimka u ah soo saarista falanqaynta ELISA.
9.1.4 Amadhan iftiinka oo dabool microwells inta lagu jiro soo saarista.
9.2 Tallaabooyinka Qiimaynta
9.2.1 Ka saar dhammaan reagents heerkulka qolka (20-25 ℃) in ka badan 30 daqiiqo, si tartiib ah u rux ka hor isticmaalka.
9.2.2 Soo saar microwells-ka loo baahan yahay oo ku soo celi inta soo hadhay bacda qufulka 2-8℃ isla markiiba.
9.2.3 Xalka dhaqidda la qasi jiray waa in dib loo diiriyaa si uu ugu jiro heerkulka qolka ka hor inta aan la isticmaalin.
9.2.4Tirada:La tiriyey boos kasta oo microwell ah iyo dhammaan heerarka iyo muunadaha waa in lagu socodsiiyaa si nuqul ah.Diiwaangeli jaangooyooyinka iyo tusaalooyinka boosaska.
9.2.5Add xal caadi ah / muunad iyo xal antibody: Ku dar 50µl oo xal caadi ah ((xirmo la siiyay)) ama muunad loo diyaariyey ceelasha u dhigma.Ku dar 50µl oo ah xal antibody ah (xirmo la siiyay).Si tartiib ah isku qas adigoo gacanta ku ruxaya saxanka oo ku dabool 30min at 37℃ dabool.
9.2.6Dhaq: Si tartiib ah uga saar daboolka oo nadiifi dareeraha ceelasha oo ku raaci microwells-ka 250µl xal dhaqid oo la badhxay (xal3) inta u dhaxaysa 10s ilaa 4-5 jeer.Biyaha haraaga ku nuugo warqad nuugista (xumbada hawada inteeda kale waxaa lagu baabi'in karaa caarad aan la isticmaalin).
9.2.7Ku dar enzyme conjugate: Ku dar 100ml oo ah xal conjugate enzyme (xirmo la siiyayCeel kasta, si tartiib ah isku qas oo ku rid 30min at 37 ℃ dabool.Mar kale ku celi talaabada dhaqida.
9.2.8MidabayntaKu dar 50µl oo xal ah (xirmo la siiyayiyo 50µl oo xal ah (B)xirmo la siiyay) ceel walba.Si tartiib ah isku qas oo ku rid 15 daqiiqo at 37 ℃ dabool.
9.2.9CabbirKu dar 50µl ee xalka joogsiga (xirmo la siiyay) ceel walba.Si tartiib ah isku qas oo cabbir nuugista 450nm (Waxaa la soo jeediyay in lagu cabbiro laba-mawjadaha dhererka ee 450/630nm. Akhri natiijada 5min gudahood ka dib markaad ku darto xal joojinta).
10. Natiijooyinka
10.1 boqolkiiba nuugista
Qiimaha celceliska ee qiyamka nuugista ee loo helay jaangooyooyinka iyo shaybaarada waxaa loo qaybiyaa qiimaha nuugista ee heerka koowaad (heer eber) waxaana lagu dhuftey 100%.Heerarka eber ayaa sidaas laga dhigay 100% qiimaha nuugista waxaa lagu soo xigtay boqolkiiba.
B
Nuugista (%) = —— × 100%
B0
B —— heerka nuugista (ama muunad)
B0 —— nuugista heerka eber
10.2 Qalooca caadiga ah
---- Si loo sawiro qalooca caadiga ah: Qaado qiimaha nuugista ee jaangooyooyinka sida dhidibka y, semi logarithmic ee fiirsashada heerka tylosin ee xalka (ppb) sidii x-dhidibka.
----Xoojinta tylosin ee muunad kasta (ppb), kaas oo laga akhrin karo qalooca cabbirka, waxaa lagu dhuftaa qodobka Dilution ee u dhigma ee muunad kasta oo la raaco, waxaana la helayaa uruurinta dhabta ah ee muunadda.
Fadlan ogow:
software gaar ah ayaa loo sameeyay falanqaynta xogta, kaas oo la bixin karo haddii la codsado.
11. Dareenka, saxnaanta iyo saxnaanta
Dareenka Tijaabada:1.5ppb
Xadka ogaanshaha:
Nudaha xoolaha …………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………….1.5ppb Caano………………………………………………………………………………….15ppb Saxnimada:
Nudaha xoolaha …………………………………………………………………………………………………………………
Caani ......................................... .. .......... ...... 80 ± 10%
Saxnaanta:
Kala duwanaanshaha xirmada ELISA waa in ka yar 10%.
12. Ogaysiis
12.1 Celceliska qiyamka nuugista ee loo helay halbeegyada iyo shaybaarada waa la dhimi doonaa haddii reagents-yada iyo muunadaha aan lagu maamulin heerkulka qolka (20-25 ℃).
12.2 Ha u ogolaan microwells in ay qallalaan inta u dhaxaysa tillaabooyinka si aad uga fogaato dib-u-soo-noqod guul-darraysan oo ay ku dhaqaaqdo tallaabada xigta isla markiiba ka dib marka aad taabato haysashada microwells.
12.3 Si tartiib ah u rux reagenti kasta ka hor inta aanad isticmaalin.
12.4 Haraggaaga ka fogee xalka joogsiga ee waa 0.5MH2SO4xal.
12.5 Ha isticmaalin xirmooyinka waqtigoodii dhacay.Ha beddelin reajentiyeyaasha dufcadaha kala duwan, haddii kale waxay hoos u dhigi doontaa dareenka.
12.6 Xirmooyinka ELISA ku hay 2-8℃, ha barafaysan.Xir taarikada nasashada microwell, Ka fogow iftiinka qorraxda tooska ah inta lagu jiro dhammaan soo-baxyada.Daboolka taarikada microtiter ayaa lagula talinayaa.
12.7 Xalka substrate-ka waa in laga tagaa haddii uu midabyo.Reagents-yadu way xumaan karaan haddii qiimaha nuugista (450/630nm) ee heerka eber uu ka yar yahay 0.5 (A450nm<0.5).
12.8 Dareen-celinta midabaynta waxay u baahan tahay 15min ka dib marka lagu daro xalalka A iyo xalalka B. Waxaadna sii dheerayn kartaa wakhtiga soo-jiidashada ilaa 20min ama ka badan haddii midabku aad u fudud yahay in la go'aamiyo.Waligaa ha dhaafin 30min, taa caksigeeda, si sax ah u gaabin wakhtiga soo-jiidashada.
12.9 Heerkulka falcelinta ugu fiican waa 37 ℃.Heerkulka sare ama ka hooseeya wuxuu u horseedi doonaa isbeddelada dareenka iyo qiyamka nuugista.
13. Kaydinta
Xaaladda kaydinta: 2-8 ℃.
Muddada kaydinta: 12 bilood.
