samfur

Ƙa'idar Gwaji

Wannan kit ɗin ya dogara ne akan fasahar ELISA mai fa'ida kai tsaye.Rijiyoyin microtiter an lullube su da antigen mai hade.Flumequineragowar a cikin samfurin yana gasa tare da antigen da aka rufe akan farantin microtiter don maganin rigakafi.Bayan ƙari na enzyme mai lakabi anti-antibody, ana amfani da substrate na TMB don nuna launi.Shanye samfurin yana da alaƙa da alaƙa da tetracycline da ke zaune a ciki, bayan kwatanta da Standard Curve, wanda aka ninka ta hanyar dilution mahara, ana iya ƙididdige yawan ragowar Flumequine a cikin samfurin.

Aikace-aikace

Ana iya amfani da wannan kit ɗin a cikin ƙididdigewa da ƙididdiga na ƙimar flumequine a cikin zuma.

Maganganun ra'ayi

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

Abubuwan da ake buƙata

Kayan aiki

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

┅┅Homogenizer ko mai ciki

┅┅ Shaker

┅┅ Vortex Mixer

┅┅Centrifuge

┅┅ Ma'aunin nazari (inductance: 0.01g)

┅┅ Pipette da aka sauke: 15ml

┅┅ Rubber pipette kwan fitila

┅┅ Polystyrene Centrifuge tube: 15ml, 50ml

┅┅ Gilashin gwajin tube: 10ml

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

250 ml - multipipette

Reagents

┅┅n-hexane (AR)

┅┅ Methylene chloride (AR)

┅┅Acetonitrile (AR)

┅┅ Ruwan da ba a so

---Hydrochloric acid (AR)

Abubuwan Kit

● Farantin Microtiter tare da rijiyoyin 96 da aka rufe da antigen

● Daidaitaccen mafita (kwalabe 6 × 1ml / kwalban)

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

● Babban kulawa daidaitaccen tsari: (1ml / kwalban)

…………………………………………………………………………100ppb

● Enzyme conjugate 12ml………………………………. jan hula

● Maganin antibody 7ml ………………………………………………….. koren hula

● Magani A 7ml………………………………………………….. farar hula

● Magani B 7ml ……………………………………………………. ja hula

● Tsaida bayani 7ml …………………………………………………

● 20XMaganin wankin ƙwanƙwasa 40ml

……………………………………………………………………

●2X Maganin cirewa 50ml……………………………… shudi hula

Shirye-shiryen Reagents

7.1 Samfurin zuma

Magani 1: 0.2 M Hydrochloric acid bayani

Nauyin 41.5ml Mai daɗaɗɗen hydrochloric acid, tsarma da ruwa mai narkewa zuwa 500 ml.

Magani 2: Maganin wankewa

Tsarma da mayar da hankali wanke bayani da deionized ruwa a cikin girma rabo na 1:19, wanda za a yi amfani da wanke faranti.da diluted bayani za a iya adana a 4 ℃ ga 1 watan.

Magani3: maganin cirewa

Tsarma da 2 × mai da hankali cire bayani tare da deionized ruwa a cikin girma rabo na 1: 1 (ko dogara da abin da ake bukata), wanda za a yi amfani da samfurin hakar.Wannan diluted bayani za a iya kiyaye ga 1 watan a 4 ℃.

Misali Shirye-shiryen

8.1 Sanarwa da kariya ga masu amfani kafin aiki

(a) Da fatan za a yi amfani da nasihu guda ɗaya a cikin aiwatar da gwaji, kuma canza tukwici lokacin shan reagent daban-daban.

(b) Tabbatar cewa duk kayan aikin gwaji suna da tsabta , in ba haka ba zai haifar da sakamakon binciken.

8.2Samfurin zuma

--Nuna 2g± 0.05g samfurin zuma a cikin bututun polystyrene centrifuge 50ml,

----A saka 2ml 0.2M Hydrochloric acid solution (Solution 1), vortex a gauraya shi gaba daya, sannan a zuba 8ml methylene chloride, a girgiza da shaker na 5min don narkar da shi gaba daya;

--Centrifuge na 10 min, aƙalla 3000g a dakin da zafin jiki (20-25 ℃);

--- Cire lokaci mai girma, ɗauki 2 ml na maganin kwayoyin halitta na substrate zuwa bututun gilashin 10 ml. bushe yankin ƙarƙashin ruwan wanka na Nitrogen kwarara (50-60 ℃)

----A ƙara 1 ml n-hexane, vortex don 30s, sannan ƙara 1ml cirewar maganin (maganin 3), sake juyi na 1min.Centrifuge na 5min, aƙalla 3000g a dakin da zafin jiki (20-25 ℃);

--- Cire lokaci mai girma, ɗauki 50ml don tantancewa;

9. Tsarin tantancewa

9.1 Sanarwa kafin tantancewa

9.1.1 Tabbatar cewa duk reagents da microwells duk suna cikin zafin jiki (20-25 ℃).

9.1.2 Koma duk sauran reagents zuwa 2-8 ℃ nan da nan bayan amfani.

9.1.3 Wanke microwells daidai wani muhimmin mataki ne a cikin tsarin tantancewa;shine muhimmin mahimmanci ga maimaitawar binciken ELISA.

9.1.4 Ka guji hasken kuma rufe microwells yayin shiryawa.

9.2 Matakan tantancewa

9.2.1 Ɗauki duk reagents a cikin zafin jiki (20-25 ℃) don fiye da 30min, homogenize kafin amfani.

9.2.2 Samo microwells da ake buƙata kuma mayar da sauran cikin jakar kulle-kulle a 2-8 ℃ nan da nan.

9.2.3 Maganin wankewar da aka diluted ya kamata a sake sanya shi a cikin zafin jiki kafin amfani.

9.2.4Lamba:Lamba kowane matsayi na microwell kuma duk ma'auni da samfurori yakamata a gudanar da su cikin kwafi.Yi rikodin ma'auni da samfurin matsayi.

9.2.5Ƙara daidaitaccen bayani/samfurin:Ƙara 50 µl na daidaitaccen bayani ko samfurin da aka shirya zuwa rijiyoyin da suka dace.Ƙara maganin antibody 50µl.Mix a hankali ta hanyar girgiza farantin da hannu kuma a sanya shi tsawon minti 30 a 25 ℃ tare da murfin.

9.2.6A wanke:Cire murfin a hankali kuma tsaftace ruwan daga cikin rijiyoyin kuma kurkura microwells tare da 250µl diluted maganin wanki (mafini 2) a tazara na 10s na sau 4-5.Shaye ragowar ruwan da takarda mai shayarwa (sauran kumfa na iska za a iya kawar da shi tare da tip mara amfani).

9.2.8.Enzyme conjugate:Ƙara maganin conjugate enzyme 100ml zuwa kowace rijiyar, Mix a hankali ta hanyar girgiza farantin da hannu kuma a sanya shi na 30min a 25 ℃ tare da murfin.Maimaita matakin wankewa kuma.

9.2.8Launi:Ƙara 50µl bayani A da 50µl bayani B a kowace rijiya.Mix a hankali ta hanyar girgiza farantin da hannu kuma a sanya shi na minti 15 a 25 ℃ tare da murfin (duba 12.8).

9.2.9Auna:Ƙara 50µl maganin tasha a kowace rijiya.Mix a hankali ta hanyar girgiza farantin da hannu kuma auna abin sha a 450nm akan sararin iska (An ba da shawarar ma'auni tare da dual-wavelength na 450/630nm. Karanta sakamakon a cikin 5min bayan ƙarin bayani tasha. ) (Za mu iya kuma auna ta gani. ba tare da tasha bayani a takaice na ELIASA kayan aiki)

Sakamako

10.1 kashi na sha

Ma'anar ma'auni na ƙimar ƙimar da aka samu don ma'auni da samfurori an raba su ta hanyar ƙimar ƙimar ma'aunin farko (misali sifili) kuma an ninka ta 100%.Ma'aunin sifili don haka ana yin daidai da 100% kuma ana ƙididdige ƙimar sha cikin kashi dari.

Abun ciki (%) = B/B0 × 100%

B ——ma'aunin sha (ko samfurin)

B0 ——shar da sifili misali

10.2 Standard Curve

Don zana madaidaicin lanƙwasa: Ɗauki ƙimar ma'auni azaman y-axis, Semi logarithmic na maida hankali na ma'aunin flumequine (ppb) azaman x-axis.

--- Aflumequinemaida hankali na kowane samfurin (ppb), wanda za'a iya karantawa daga madaidaicin daidaitawa, an ninka ta hanyar dilution mahara na kowane samfurin da aka biyo baya, kuma ana samun ainihin ƙaddamar da samfurin.

Don rage bayanai na kayan aikin ELISA, an ƙirƙira software na musamman, wanda za'a iya bayarwa akan buƙata.

11. Hankali, daidaito da daidaito

Gwajin Hankali:0.3pb ku

Samfurin Ruwan Ruwan Ruwa: 2

Iyakar ganowa

Samfurin zuma --------------------------------- -1ppb

Daidaito

Samfurin zuma ------------------------------------------------- 90±20 %

Daidaitawa

Bambance-bambancen adadin kayan aikin ELISA bai wuce 10%.

12. Sanarwa

12.1 Ma'anar ƙimar ƙimar ƙimar da aka samu don ma'auni kuma samfuran za a rage su idan ba a daidaita reagents da samfuran zuwa zafin jiki ba (20-25 ℃).

12.2 Kada ku ƙyale microwells su bushe tsakanin matakai don guje wa maimaita rashin nasara kuma kuyi aiki na gaba gaba nan da nan bayan danna mariƙin microwells.

12.3.Homogenize kowane reagents kafin amfani.

12.4.Ka nisantar da fata daga maganin tasha domin ita ce 2M H₂SO₄mafita.

12.5 Kada ku yi amfani da kayan aikin da suka wuce.Kada a musanya reagents na batches daban-daban, domin zai sauke hankali.

12.6 Yanayin ajiya:

Ajiye kayan ELISA a 2-8 ℃, kar a daskare.Rufe faranti na microwell na hutawa Ka guje wa hasken rana kai tsaye a duk lokacin da ake shiryawa.Ana ba da shawarar rufe faranti na microtiter.

12.7 Alamu ga reagents na faruwa mara kyau:

Ya kamata a watsar da maganin substrate idan ya juya launuka.

Reagents na iya zama mara kyau idan ƙimar ɗaukar abu (450/630nm) na ma'aunin sifili bai wuce 0.5 (A450nm ~ 0.5).

12.8 Yanayin launi yana buƙatar 15min bayan ƙara Magani A da Magani B. Kuma zaka iya tsawaita lokacin shiryawa daga 20min zuwa ƙari idan launi ya yi haske sosai don ƙayyade.Kada ku wuce minti 25, akasin haka, rage lokacin shiryawa yadda ya kamata.

12.9 Mafi kyawun zafin jiki shine 25 ℃.Mafi girma ko ƙananan zafin jiki zai haifar da canje-canje na hankali da ƙimar sha.

13. Adana

Yanayin ajiya: 2-8 ℃.

Lokacin ajiya: watanni 12.