alaabta

Mabaadi'da Imtixaanka

Xirmadan waxay ku salaysan tahay tignoolajiyada ELISA ee aan tooska ahayn ee tartanka.Ceelasha microtiter-ka waxaa lagu dahaadhay antigen-ka isku-xidha.FlumequineHadhaaga muunada waxay la tartameysaa antigen-ka ku dahaaran 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 tetracycline-ka ay ku dhex nooshahay, ka dib marka la barbar dhigo Standard Curve, oo lagu dhufto isku dar ah, tirada haraaga Flumequine ee muunada waa la xisaabin karaa.

Codsiyada

Qalabkan waxa loo isticmaali karaa tiro ahaan iyo falanqaynta tayada leh ee hadhaaga flumequine ee malabku.

Falcelin-celin

Flumequine ………………………………………………………………………… 100%

Qalabka Loo Baahan Yahay

Qalabka

┅┅ Mikrotiter saxan spectrophotometer (450nm/630nm)

┅┅Homogenizer ama caloosha

┅┅Shaker

┅┅ Isku darka Vortex

┅┅Centrifuge

┅┅ Isku dheelitirnaanta falanqaynta (inductance: 0.01g)

┅┅ pipette qalin jabiyay: 15ml

┅┅ Nalka pipette caag ah

┅┅Tuubada Centrifuge Polystyrene: 15ml, 50ml

┅┅ Tuubada tijaabada muraayadda: 10ml

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

250ml -multipipette

Reagents

┅┅n-hexane(AR)

┅┅Methylene chloride (AR)

┅┅Acetonitrile (AR)

┅┅Biyaha aan la karin

---Hydrochloric acid (AR)

Qaybaha Kit

● Saxan yar oo leh 96 ceel oo lagu dahaadhay antigen

● xalalka caadiga ah (6 dhalo × 1ml/dhalo)

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

● Xakamaynta heerka feejignaanta sare: (1ml/ dhalo)

…………………………………………………………100ppb

● Enzyme conjugate 12ml………………………………

Xalka difaaca jirka 7ml …………………………………………….. koofiyada cagaaran

● Xalka A 7ml………………………………………………………….. Koofi cad

● Xalka B 7ml ………………………………………………………

● Jooji xal 7ml …………………………………………………

20X Xalka dhaqidda ee xoogga leh 40ml

……………………………………………………. koofiyad hufan

●2X Xalka Soo saarista 50ml………………………………

Diyaarinta Reagents

7.1 Muunada Malabka

Xalka 1: 0.2 M Hydrochloric acid

Miisaanka 41.5ml Hydrochloric acid oo ku urursan, ku mil biyo la miiray ilaa 500 ml.

Xalka 2: Xalka dhaq

Ku qas xal dhaqidda xoogga leh biyo deionized ee saamiga mugga 1:19, kaas oo loo isticmaali doono dhaqidda taarikada.xalka la qasi karo waxaa lagu kaydin karaa at 4 ℃ 1 bil.

Xalka3: xal ka saarista

Ku qas xalka 2× ee uruursan ee soo saarista biyaha deionized ee saamiga mugga 1:1 (ama ku xiran shuruudaha), kaas oo loo isticmaali doono soo saarista muunada.Xalkan la qasi karo waxaa lagu kaydin karaa 1 bil at 4 ℃.

Tusaalaha Diyaarinta

8.1 Ogeysiiska iyo taxaddarrada isticmaaleyaasha 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 qalabka tijaabada ahi ay nadiif yihiin, haddii kale waxay saamaynaysaa natiijada qiimaynta.

8.2Muunad malabka

--Miisaan 2g± 0.05g muunada malab galay tube centrifuge 50ml polystyrene,

---Ku dar 2ml 0.2 M Hydrochloric acid (Xalka 1), vortex si aad si buuxda isugu walaaqdo, dabadeed ku dar 8ml methylene chloride, rux ruxul ilaa 5min si aad si buuxda u milmaan;

--Centrifuge 10 daqiiqo, ugu yaraan 3000g heerkulka qolka (20-25 ℃);

--Ka saar wejiga sare, qaado 2 ml oo ah substrate-ka dareeraha organic ilaa tuubo galaas 10 ml ah. qallaji substate hoostiisa qubeyska biyaha ee socodka Nitrogen (50-60℃)

-----Ku dar 1 ml n-hexane, vortex ilaa 30s, ka dibna ku dar 1ml xalinta soo saarista (xalka 3) , marlabaad 1min.Centrifuge 5min, ugu yaraan 3000g heerkulka qolka (20-25 ℃);

--Ka saar wajiga sare, qaado 50ml si aad u tijaabiso;

9. Habka qiimaynta

9.1 Ogaysii ka hor inta aan qiimayn

9.1.1 Hubi in dhammaan reagen-yada iyo microwells ay dhamaantood ku jiraan heerkulka qolka (20-25 ℃).

9.1.2 Ku soo celi dhammaan reagen-yaasha soo hadhay 2-8 ℃ isla markiiba ka dib marka la isticmaalo.

9.1.3 Si sax ah u dhaqida ceelasha yaryar waa tallaabo muhim ah oo geedi socodka qiimaynta ah;waa qodobka muhiimka u ah soo noqnoqoshada falanqaynta ELISA.

9.1.4 Ka fogow iftiinka oo dabool microwells inta lagu jiro soo-baxa.

9.2 Tallaabooyinka Qiimaynta

9.2.1 Ka saar dhammaan reagennada heerkulka qolka (20-25 ℃) in ka badan 30min, isku mid ka hor inta aanad isticmaalin.

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:Tiri meel kasta oo microwell ah iyo dhammaan heerarka iyo muunadaha waa in lagu socodsiiyaa si nuqul ah.Diiwaangeli jaangooyooyinka iyo tusaalooyinka boosaska.

9.2.5Ku dar xal/ muunad caadi ah:Ku dar 50 µl oo xal caadi ah ama muunad diyaarsan ceelasha u dhigma.Ku dar 50µl xal antibody.Si tartiib ah isku qas adigoo gacanta ku ruxaya saxanka oo ku dabool 30min at 25℃ dabool.

9.2.6Dhaq:Daboolka si tartiib ah uga saar oo ka nadiifi dareeraha ceelasha oo ku raaci microwells 250µl xal dhaqid oo la qasiday (xalka 2) 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).

9.2.8.Enzyme conjugate:Ku dar 100 ml oo ah xalka enzyme conjugate ceel kasta.Mar kale ku celi talaabada dhaqida

9.2.8Midabaynta:Ku dar 50µl xal A iyo 50µl xal B ah midkiiba.Si tartiib ah ugu qas adiga oo gacanta ku ruxaya saxanka oo ku dabool 15 daqiiqo at 25℃ dabool (eeg 12.8).

9.2.9Cabbir:Ku dar 50µl ee xalka joogsiga ceel kasta.Si tartiib ah isku qas adigoo gacanta ku ruxaya saxanka oo cabbir nuugista 450nm marka loo eego hawo bannaan (Waxaa lagu talinayaa in lagu cabbiro dhererka laba-wavelength ee 450/630nm. U akhri natiijada 5min gudahood ka dib marka lagu daro xalka joojinta.) (Waxaan sidoo kale cabbiri karnaa aragtida. iyada oo aan la joojin xalinta qalabka ELIASA)

Natiijooyinka

10.1 boqolkiiba nuugista

Qiimaha celceliska ee qiyamka nuugista ee loo helay halbeegyada iyo shaybaarrada 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.

nuugista (%) = B/B0 ×100%

B —— heerka nuugista (ama muunad)

B0 —— nuugista heerka eber

10.2 Qalooca caadiga ah

Si loo sawiro qalooca caadiga ah: U qaado qiimaha nuugista ee jaangooyooyinka sida dhidibka y, semi logarithmic ee fiirsashada heerka flumequine xalinta (ppb) sidii dhidibka x.

--- Theflumequinefiirsashada muunad kasta (ppb), kaas oo laga akhrin karo qalooca cabbirka, waxaa lagu dhuftaa dhuxusha u dhiganta ee muunad kasta oo la raaco, waxaana la helayaa uruurinta dhabta ah ee muunadda.

Si loo yareeyo xogta xirmooyinka ELISA, software gaar ah ayaa la sameeyay, kaas oo la bixin karo haddii la codsado.

11. Dareenka, saxnaanta iyo saxnaanta

Tijaabi dareenka：0.3ppb

Tusaalaha Malabka Qodobbada lagu qasi karo: 2

Xadka ogaanshaha

Muunad Malabka --------------------------------- -1ppb

Saxnaanta

Muunad malabka --------------------------------------------------- 90±20 %

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 looga fogaado soo noqnoqoshada aan lagu guulaysan oo ku dhaqaaq tallaabada xigta isla markiiba ka dib marka aad taabato haysashada microwells.

12.3.Isku soo wada duub reagenti kasta ka hor inta aanad isticmaalin.

12.4.Maqaarkaaga ka fogee xalka joogsiga sababtoo ah waa 2M H₂SO₄xal.

12.5 Ha isticmaalin xirmooyinka waqtigoodii dhacay.Ha beddelin reajentiyeyaasha dufcadaha kala duwan, waayo waxay hoos u dhigi doontaa dareenka.

12.6 Xaaladda kaydinta:

Xirmooyinka ELISA ku hay 2-8℃, ha barafaysan.Xir nasashada taarikada microwell Ka fogow iftiinka qorraxda tooska ah inta lagu jiro dhammaan soo-baxyada.Daboolka taarikada microtiter ayaa lagula talinayaa.

12.7 Tilmaamaha reagents-ku xumaaday:

Xalka substrate-ka waa in laga tagaa haddii uu midabyo isu rogo.

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 markaad ku darto Xalka A iyo Xalka B. Oo waxaad sii dheerayn kartaa wakhtiga soo noqnoqda inta u dhaxaysa 20min ilaa in ka badan haddii midabku aad u fudud yahay in la go'aamiyo.Weligaa ha dhaafin 25min, Taa beddelkeeda, si habboon u gaabin wakhtiga soo-jiidashada.

12.9 Heerkulka falcelinta ugu fiican waa 25 ℃.Heerkulka sare ama ka hooseeya wuxuu u horseedi doonaa isbeddelada dareenka iyo qiyamka nuugista.

13. Kaydinta

Xaaladda kaydinta: 2-8 ℃.

Muddada kaydinta: 12 bilood.