Gasar Enzyme Immunoassay Kit don Ƙididdigar Ƙididdigar Tylosin
Gasar Enzyme Immunoassay Kit don
Ƙididdigar ƙididdiga naTylosin
1. Fage
TylosinKwayoyin cuta ne na macrolide, wanda aka fi amfani dashi azaman antibacterial da anti-mycoplasma.An kafa tsauraran MRLs tun lokacin da wannan magani na iya haifar da mummunan sakamako a wasu ƙungiyoyi.
Wannan kit ɗin sabon samfuri ne wanda ya dogara da fasahar ELISA, mai sauri, mai sauƙi, daidai kuma mai kulawa idan aka kwatanta da bincike na kayan aiki gama gari kuma yana buƙatar sa'o'i 1.5 kawai a cikin aiki ɗaya, yana iya rage girman kuskuren aiki da ƙarfin aiki.
2. Ka'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.Ragowar Tylosin a cikin samfurin yana gasa tare da antigen da aka lullube akan farantin microtiter don maganin rigakafi.Bayan ƙari na enzyme mai lakabi anti-antibody, ana amfani da substrate na TMB don nuna launi.Shawarar samfurin yana da alaƙa da alaƙa da tylosin da ke zaune a ciki, bayan kwatanta da Standard Curve, ninka ta hanyar dilution factor, ana iya ƙididdige yawan ragowar tylosin a cikin samfurin.
3. Aikace-aikace
Ana iya amfani da wannan kit ɗin a ƙididdigewa da ƙididdige ƙimar tylosin a cikin nama na dabba (kaza, naman alade, agwagwa) da madara, zuma, kwai, da sauransu.
4. Matsala-matsayi
Tylosin………………………………………………………………………………………………………
Tilmicosin…………………………………………………………………………………………………………
5. Abubuwan da ake buƙata
5.1 Kayan aiki:
----Microtiter farantin spectrophotometer (450nm/630nm)
---- Rotary evaporator ko kayan bushewar nitrogen
----homogenizer
----Shakar
----Centrifuge
---- Ma'aunin nazari (inductance: 0.01g)
----Piette da aka sauke: 10ml
---- Rubber pipette kwan fitila
----Filin girma: 10ml
----Polystyrene centrifuge bututu: 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)
---- Ruwan da aka lalata
6. Abubuwan Kit
l Microtiter farantin tare da 96 rijiyoyin rufi da antigen
l Standard mafita (5 kwalabe, 1ml / kwalban)
0ppb, 0.5ppb, 1.5ppb, 4.5ppb, 13.5ppb
l Daidaitaccen sarrafawa: (1ml/kwalba)1ppm ku
l Enzyme conjugate 1ml………………………………………
l Maganin antibody 7ml………………………………………
l Magani A 7ml…………………………………………………
l Magani B 7ml………………………………………………. ja hula
l Tsayar da maganin 7ml.………………………………. rawaya hula
l 20 × maida hankali wanke bayani 40ml
……………………………………………………
l 4 × mai da hankali hakar bayani 50ml
………………………………………………
7. Shirye-shiryen Reagent:
Magani 1:0.1mol/L NaOH bayani
Auna 0.4g NaOH zuwa 100ml ruwan da aka lalatar kuma a gauraya gaba daya.
Magani 2: 1mol/L NaOH bayani
Auna 4g NaOH zuwa 100ml ruwan da aka lalatar kuma a gauraya gaba daya.
Magani 3: Carbonate buffer gishiri
Magani1: 0.2M PB
Narkar da 51.6g na Na2HPO4· 12H2O, 8.7g na NaH2PO4· 2H2O tare da deionized ruwa kuma tsarma zuwa 1000ml.
Magani2: Maganin cirewa
Tsarma da 2 × mayar da hankali hakar bayani tare da deionized ruwa a cikin girma rabo na 1: 1 (misali 10ml na 2 × hakar bayani + 10ml na deionized ruwa), wanda za a yi amfani da shi don hakar samfurin,wannan bayani za a iya adana a 4 ℃ ga wata 1.
Magani3: Maganin wanka
Tsarma da 20 × mayar da hankali bayani wanke tare da deionized ruwa a cikin girma rabo na 1:19 (misali 5ml na 20× wankin bayani + 95ml na deionized ruwa), wanda za a yi amfani da shi don wanke faranti.Wannan bayani za a iya adana a 4 ℃ ga wata 1.
8. Misali Shirye-shiryen
8.1 Sanarwa da kariya 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 suna da tsabta.
(c) Ajiye samfurin nama a daskare.
(d) Ya kamata a yi amfani da samfurin da aka shirya don tantancewa lokaci guda.
8.2 Naman dabba (kaza, naman alade, da sauransu)
---- Haɗa samfurin tare da homogenizer;
----Dauki 2.0 ± 0.05g na homogenate a cikin 50ml polystyrene centrifuge tube;ƙara 2 ml na 0.2M PB (mafita1) , girgiza don narkewa, sa'an nan kuma ƙara 8ml na ethyl acetate kuma girgiza sosai don 3min;
----Centrifuge don rabuwa: 3000g / yanayin zafi / 5min.
---- Canja wurin 4ml na mafi girman yanayin halitta a cikin bututun gilashin 10ml, bushe tare da ruwan wanka na 50-60 ℃ a ƙarƙashin rafin iskar gas;
----Narkar da busassun ragowar tare da 1ml na n-hexane, vortex don 30s don narkewa, sannan ƙara 1ml na maganin cirewa (mafita2), vortex na 1 min.centrifuge don rabuwa: 3000g / yanayin zafi / 5min
---- Cire mafi girman lokaci n-hexane;Ɗauki 50 μl na lokaci mai ruwa mai ruwa don tantancewa.
Matsalolin dilution: 1
8.2 Madara
----Dauki 100μl na danyen samfurin madara, haxa tare da 900μl na maganin cirewa (mafita2), da kuma Mix gaba daya.
---- Ɗauki 50μl na maganin da aka shirya don tantancewa.
Matsalolin dilution: 10
9. Tsarin tantancewa
9.1 Sanarwa kafin tantancewa
9.1.1Tabbatar cewa duk reagents da microwells duk suna cikin zafin jiki (20-25 ℃).
9.1.2Mayar da duk sauran reagents zuwa 2-8℃nan da nan bayan amfani.
9.1.3Wanke microwells daidai mataki ne mai mahimmanci a cikin aikin tantancewa;Yana da mahimmancin mahimmanci ga sake fasalin binciken ELISA.
9.1.4 Aɓata hasken kuma rufe microwells yayin shiryawa.
9.2 Matakan tantancewa
9.2.1 Cire duk reagents a cikin zafin jiki (20-25 ℃) fiye da minti 30, girgiza a hankali 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:An ƙididdige kowane matsayi na microwell kuma duk ƙa'idodi da samfurori yakamata a gudanar da su cikin kwafi.Yi rikodin ma'auni da samfurin matsayi.
9.2.5Add daidaitaccen bayani/samfurin da maganin antibody: Ƙara 50µl na daidaitaccen bayani (((kit ya bayar)) ko shirya samfurin zuwa rijiyoyin da suka dace.Ƙara 50µl na maganin antibody (kit ya bayar).Mix a hankali ta hanyar girgiza farantin da hannu kuma a sanya shi tsawon minti 30 a 37 ℃ tare da murfin.
9.2.6Wanka: Cire murfin a hankali kuma tsaftace ruwan daga cikin rijiyoyin kuma kurkura microwells tare da 250µl diluted wanke bayani (mafita3) a tazara na 10s na 4-5times.Shaye ragowar ruwan da takarda mai shayarwa (sauran kumfa na iska za a iya kawar da shi tare da tip mara amfani).
9.2.7Ƙara enzyme conjugate: Ƙara 100ml na maganin conjugate enzyme (kit ya bayar) ga kowace rijiya, a haxa a hankali kuma a saka tsawon 30min a 37 ℃ tare da murfin.Maimaita matakin wankewa kuma.
9.2.8Launi: Ƙara 50µl na maganin A(kit ya bayarda kuma 50µl na maganin B (kit ya bayar) ga kowace rijiya.Mix a hankali kuma a dafa don mintina 15 a 37 ℃ tare da murfin.
9.2.9Auna: Ƙara 50µl na maganin tasha (kit ya bayar) ga kowace rijiya.Mix a hankali kuma auna abin sha a 450nm (An ba da shawarar auna tare da dual-wavelength na 450/630nm. Karanta sakamakon a cikin 5min bayan ƙara bayani tasha).
10. Sakamako
10.1 kashi na sha
Ma'anar ma'auni na ƙimar ƙimar da aka samo don ma'auni da samfurori an raba su ta hanyar ƙimar ƙimar ƙimar 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.
B
Abun sha (%) = —— × 100%
B0
B ——ma'aunin sha (ko samfurin)
B0 ——shar da sifili misali
10.2 Standard Curve
----Don zana daidaitaccen lanƙwasa: Ɗauki ƙimar ma'auni azaman y-axis, Semi logarithmic na ƙaddamar da ma'aunin ma'aunin tylosin (ppb) azaman x-axis.
---- Matsakaicin tylosin na kowane samfurin (ppb), wanda za'a iya karantawa daga ma'auni na calibration, an ninka ta hanyar daidaitattun Dilution factor na kowane samfurin da aka biyo baya, kuma ana samun ainihin ƙaddamar da samfurin.
Da fatan za a lura:
an ƙera software na musamman don nazarin bayanai, waɗanda za a iya bayar da su akan buƙata.
11. Hankali, daidaito da daidaito
Gwajin Hankali:1.5ppb
Iyakar ganowa:
Naman dabba …………………………………………………………………………………………………………………… 1.5ppb Milk .........................................................15ppb Daidaito:
Naman dabba……………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………………… 80±15%
Milk ........................................................... ......... ... 80 ± 10%
Daidaito:
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 ka ƙyale microwells su bushe tsakanin matakai don kauce wa sake haifar da rashin nasara da kuma aiki mataki na gaba nan da nan bayan danna mariƙin microwells.
12.3 Girgiza kowane reagent a hankali kafin amfani.
12.4 Ka nisanta fatar jikinka daga maganin tasha domin ita ce 0.5MH2SO4mafita.
12.5 Kada ku yi amfani da kayan aikin da suka wuce.Kada a musanya reagents na batches daban-daban, in ba haka ba zai sauke hankali.
12.6 Rike kayan ELISA a 2-8 ℃, kar a daskare.Rufe faranti na microwell, guje wa hasken rana kai tsaye yayin duk abubuwan da suka faru.Ana ba da shawarar rufe faranti na microtiter.
12.7 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 Halin launi yana buƙatar 15min bayan ƙara bayani A da bayani B. Kuma za ku iya tsawaita lokacin shiryawa zuwa 20min ko fiye idan launi ya yi haske sosai don ƙayyade.Kada ku wuce minti 30, akasin haka, rage lokacin shiryawa yadda ya kamata.
12.9 Mafi kyawun zafin jiki shine 37 ℃.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.
