sehlahisoa

Competitive Enzyme Immunoassay Kit for Quantitative Analysis of Tylosin

Tlhaloso e Khutšoanyane:

Tylosin ke lithibela-mafu tsa macrolide, tse sebelisoang haholo-holo e le antibacterial le anti-mycoplasma.Li-MRL tse thata ha li e-s'o thehoe kaha moriana ona o ka lebisa litla-morao tse tebileng lihlopheng tse itseng.

Sehlahisoa sena ke sehlahisoa se secha se ipapisitseng le theknoloji ea ELISA, e potlakile, e bonolo, e nepahetse ebile e na le kutloelo-bohloko ha e bapisoa le tlhahlobo e tloaelehileng ea lisebelisoa mme e hloka lihora tse 1.5 ts'ebetsong e le 'ngoe, e ka fokotsa phoso ea ts'ebetso le matla a ts'ebetso haholo.


Lintlha tsa Sehlahisoa

Li-tag tsa Sehlahisoa

Competitive Enzyme Immunoassay Kit bakeng sa

Quantitative Analysis eaTylosin


1. Semelo

Tylosinke lithibela-mafu tsa macrolide, tse sebelisoang haholo-holo joalo ka antibacterial le anti-mycoplasma.Li-MRL tse thata ha li e-s'o thehoe kaha moriana ona o ka lebisa litla-morao tse tebileng lihlopheng tse itseng.

Sehlahisoa sena ke sehlahisoa se secha se ipapisitseng le theknoloji ea ELISA, e potlakile, e bonolo, e nepahetse ebile e na le kutloelo-bohloko ha e bapisoa le tlhahlobo e tloaelehileng ea lisebelisoa mme e hloka lihora tse 1.5 ts'ebetsong e le 'ngoe, e ka fokotsa phoso ea ts'ebetso le matla a ts'ebetso haholo.

2. Molao-motheo oa Teko

Setsi sena se ipapisitse le theknoloji e sa tobang ea tlholisano ea ELISA.Liliba tsa microtiter li koahetsoe ka li-antigen tse kopanyang.Masalla a tylosin sampole a qothisana lehlokoa le antigen e tlositsoeng holim'a poleiti ea microtiter bakeng sa lesole la 'mele.Kamora ho eketsoa ha enzyme e ngotsoeng anti-anti-antibody, substrate ea TMB e sebelisoa ho bonts'a 'mala.Ho tlosoa ha sampuli ho amana hampe le tylosin e lulang ho eona, ka mor'a ho bapisa le Standard Curve, e atolositsoeng ke ntlha ea dilution, palo ea masala a tylosin ka sampuli e ka baloa.

3. Likopo

Sesebelisoa sena se ka sebelisoa tlhahlobong ea bongata le ea boleng ba masala a tylosin liphatseng tsa liphoofolo (khoho, nama ea kolobe, letata) le lebese, mahe a linotsi, lehe, joalo-joalo.

4. Liphetoho tse fapaneng

Tylosin………………………………………………………..100%

Tilmicosin…………………………………………………<2%

5. Lintho Tse Hlokehang

5.1 Thepa:

----- Microtiter plate spectrophotometer (450nm/630nm)

----Sesebediswa sa rotary evaporator kapa nitrogen drying

---- homogenizer

---- Shaker

-----Setsi

----Analytical balance (inductance: 0.01g)

-----Pipette e fumaneng mangolo: 10ml

----Lebone la peipi ea rabara

---- Botlolo ea Volumetric: 10ml

-----Polystyrene centrifuge tubes: 50ml

----Li-Micropipettes: 20-200ml, 100-1000ml

250ml-multipipette

5.2 Lisebelisoa:

----Sodium hydroxide (NaOH, AR)

----Sodium bicarbonate (NaHCO3,AR)

---- Sodium carbonate (NaCO3, AR)

----Trichloroacetic acid (AR)

---- Acetonitrile (AR)

---- Ethyl acetate (AR)

┅┅n-Hexane (AR)

-----Metsi a hloekisitsoeng

6. Likaroloana tsa Kit

l Letlapa la Microtiter le liliba tse 96 tse koahetsoeng ka antigen

l Litharollo tse tloaelehileng (libotlolo tse 5, 1ml/botlolo)

0ppb, 0.5ppb, 1.5ppb, 4.5ppb, 13.5ppb

l Taolo e tloaelehileng ea Spiking: (1ml/botlolo)1ppm

l Enzyme conjugate 1ml ……………..…..…kepisi e khubelu

l Tharollo ea Antibody 7ml ……………….……kepisi e tala

l Tharollo A 7ml …………………….….……kepisi e tšoeu

l Tharollo B 7ml…………………………………..kepisi e khubelu

l Stop solution 7ml. ……………….……….yellow cap

l 20 × tharollo e tebileng ea ho hlatsoa 40ml

……………………………………………….. transparent cap

l 4 × tharollo e tsitsitseng ea ho ntša 50ml

……………………………………………………….kepisi e putsoa

7. Tokiso ea li-reagents:

Tharollo 1:0.1mol/L tharollo ea NaOH

Beha boima ba 0.4g NaOH ho isa ho 100ml ea metsi a deionized 'me u kopanye ka ho feletseng.

Tharollo 2: 1mol/L tharollo ea NaOH

Beha boima ba 4g NaOH ho isa ho 100ml ea metsi a ntšitsoeng 'me u kopanye ka ho feletseng.

Tharollo 3: Letsoai la carbonate buffer

Tharollo1: 0.2M PB

Qhala 51.6g ea Na2HPO4·12H2O, 8.7g ea NaH2PO4·2H2O ka metsi a deionized mme o hlapolle ho 1000ml.

Tharollo2: Tharollo ea ho ntša

Hlakola tharollo ea 2 × concentrated extraction ka metsi a deionized ka tekanyo ea 1: 1 (mohlala 10ml ea 2× tharollo ea ho ntša + 10ml ea metsi a deionized), e tla sebelisoa bakeng sa ho ntša mohlala,tharollo ena e ka bolokoa ho 4 ℃ bakeng sa khoeli e le 'ngoe.

Tharollo3: Hlatsoa tharollo

Hlatsoa tharollo ea 20 × concentrated ea ho hlatsoa ka metsi a hloekisitsoeng ka tekanyo ea 1:19 (mohlala 5ml ea 20× hlatsoa tharollo + 95ml ea metsi a deionized), e tla sebelisetsoa ho hlatsoa lipoleiti.Tharollo ena e ka bolokoa ho 4 ℃ bakeng sa khoeli e le 'ngoe.

8. Mehlala ea Litokisetso

8.1 Tsebiso le litemoso pele ho ts'ebetso:

(a) Ka kopo sebelisa malebela a nako e le 'ngoe ha u ntse u etsa liteko, 'me u fetole malebela ha u amohela reagent e fapaneng.

(b) Etsa bonnete ba hore lisebelisoa tsohle li hloekile.

(c) Boloka sampole ea linama sebakeng se leqhoa.

(d) Mohlala o lokisitsoeng o lokela ho sebelisoa bakeng sa tlhahlobo hang-hang.

8.2 Lithipa tsa liphoofolo (khoho, nama ea kolobe, joalo-joalo)

---- Homogenize sampole ka homogenizer;

----Nka 2.0±0.05g ea homogenate ho 50ml polystyrene centrifuge tube;eketsa 2ml ea 0.2M PB (tharollo1) , sisinya ho qhibiliha, ebe u eketsa 8ml ea ethyl acetate 'me u sisinyehe ka matla bakeng sa 3min;

----Centrifuge bakeng sa karohano: 3000g / mocheso o teng / 5min.

----Fetisetsa 4ml ea karolo ea tlhaho e matlahali ka har'a tube ea khalase ea 10ml, e omeletseng ka 50-60 ℃ metsi a ho hlapa ka tlas'a khase ea naetrojene;

----Qholosa se setseng se ommeng ka 1ml ea n-hexane, vortex bakeng sa 30s ho qhibiliha, ebe o eketsa 1ml ea tharollo ea monko (tharollo2), vortex bakeng sa 1min.centrifuge bakeng sa karohano: 3000g / mocheso ambient / 5min

---- Tlosa mokhahlelo oa supernatant n-hexane;nka 50μl ea karolo ea metsi ea substrate bakeng sa tlhahlobo.

 

Dilution factor: 1

 

8.2 Lebese

----Nka 100μl ea sampole ea lebese le tala, kopanya le 900μl ea mokelikeli oa mokelikeli (tharollo2), 'me u kopanye ka ho feletseng.

----Nka 50μl ea tharollo e lokiselitsoeng bakeng sa tlhahlobo.

 

Dilution factor: 10

 

9. Mokhoa oa ho hlahloba

9.1 Hlokomela pele ho tlhahlobo

9.1.1Etsa bonnete ba hore li-reagents tsohle le li-microwell kaofela li mocheso oa kamore (20-25 ℃).

9.1.2Khutlisetsa li-reagents tsohle ho 2-8hang ka mora ho sebedisoa.

9.1.3Ho hlatsoa li-microwell ka nepo ke mohato oa bohlokoa ts'ebetsong ea tlhahlobo;ke ntlha ea bohlokoa ho ikatisa ha tlhahlobo ea ELISA.

9.1.4 Ahlakola leseli 'me u koahele li-microwell nakong ea ho qhoqhoa.

9.2 Mehato ea Tekolo

9.2.1 Ntša li-reagents tsohle ka mocheso oa kamore (20-25℃) ho feta 30min, sisinya ka bonolo pele u li sebelisa.

9.2.2 Ntša li-microwell tse hlokahalang 'me u khutlisetse tse ling ka mokotleng oa zip-lock ka 2-8℃ hang-hang.

9.2.3 Tharollo ea ho hlatsoa e hlapollotsoeng e lokela ho futhumatsoa hore e be mocheso oa kamore pele e sebelisoa.

9.2.4Nomoro:Nomoro ea maemo a microwell 'me litekanyetso tsohle le lisampole li lokela ho tsamaisoa ka makhetlo a mabeli.Ngola maemo le lisampole maemo.

9.2.5Add tharollo e tloaelehileng/sampole le tharollo ea antibody: Kenya 50µl ea tharollo e tloaelehileng ((kit e fanoeng)) kapa sampole e lokiselitsoeng liliba tse tsamaellanang.Kenya 50µl ea motsoako oa antibody (kit e fanoeng).Kopanya ka bonolo ka ho sisinya poleiti ka letsoho 'me u e kenye metsotso e 30 ho 37 ℃ ka sekoahelo.

9.2.6Hlatsoa: Tlosa sekoahelo ka bonolo 'me u hloekise mokelikeli ka har'a liliba' me u hlatsoe li-microwell ka tharollo e hlatsoitsoeng ea 250µl (tharollo3) ka karohano ea 10s bakeng sa 4-5 linako tse ling.Monya metsi a setseng ka pampiri e monyang (bubble e setseng ea moea e ka felisoa ka ntlha e sa sebelisoeng).

9.2.7Kenya conjugate ea enzyme: Kenya 100ml ea enzyme conjugate solution (kit e fanoeng) selibeng se seng le se seng, kopanya ka bonolo 'me u etsetse metsotso e 30 ho 37℃ ka sekoahelo.Pheta mohato oa ho hlatsoa hape.

9.2.8Mebala: Kenya 50µl ea tharollo A(kit e fanoeng) le 50µl ea tharollo B (kit e fanoeng) selibeng se seng le se seng.Kopanya ka bonolo 'me u etsetse metsotso e 15 ho 37℃ ka sekoahelo.

9.2.9Lekanya: Kenya 50µl ea tharollo ea ho emisa (kit e fanoeng) selibeng se seng le se seng.Kopanya ka bonolo 'me u lekanye monyetla ho 450nm (Ho khothaletsoa tekanyo e nang le maqhubu a mabeli a 450/630nm. Bala sephetho ka hare ho 5min ka mor'a ho eketsa tharollo ea ho emisa).

10. Liphetho

10.1 Peresente ea ho monya

Litekanyetso tse tloaelehileng tsa litekanyetso tsa ho monya tse fumanoeng bakeng sa litekanyetso le lisampole li arotsoe ke boleng ba ho monya ba tekanyo ea pele (zero standard ) 'me e atolosoa ka 100%.Tekanyetso ea zero ka hona e etsoa hore e lekane le 100% mme litekanyetso tsa ho monya li qotsitsoe ka liphesente.

B

Absorbance (%) = —— × 100%

B0

B - - absorbance standard (kapa sampole)

B0 -- absorbance zero standard

10.2 Mokokotlo o Tloaelehileng

----Ho etsa mokokotlo o tlwaelehileng: Nka boleng ba ho monya ba maemo joalo ka y-axis, semi logarithmic ea concentration ea tylosin standards solution (ppb) joalo ka x-axis.

----Mahlo a tylosin a sampole e 'ngoe le e 'ngoe (ppb), e ka baloang ho tsoa lekhalong la calibration, e atisoa ke Dilution factor e tsamaellanang ea sampole e 'ngoe le e 'ngoe e lateloang, 'me bongata ba sampole boa fumanoa.

Ka kopo hlokomela:

software e khethehileng e entsoe bakeng sa tlhahlobo ea data, e ka fanoang ka kopo.

11. Kutloelo-bohloko, ho nepahala le ho nepahala

Boikutlo ba Teko:1.5ppb

Moeli oa ho fumanoa:

Lithishu tsa liphoofolo…………………………………………………1.5ppb Lebese ………………………………………………………..15ppb Ho nepahala:

Lithipa tsa liphoofolo ………………………………………………80±15%

Lebese …………………………………………………..80±10%

Nepahalo:

Phapang coefficient ea ELISA kit e ka tlase ho 10%.

12. Hlokomela

12.1 Litekanyetso tse tloaelehileng tsa litekanyetso tsa ho monya tse fumanoang bakeng sa litekanyetso le lisampole li tla fokotsoa haeba li-reagents le sampuli li sa laoloe ho mocheso oa kamore (20-25℃).

12.2 Se ke oa lumella li-microwells ho omella lipakeng tsa mehato ho qoba ho se atlehe ho ikatisa le ho sebetsa mohato o latelang hang ka mor'a ho tlanya sets'oants'o sa li-microwell.

12.3 Tšoara reagent ka 'ngoe ka bonolo pele u e sebelisa.

12.4 Beha letlalo la hau hole le tharollo ea ho emisa hobane ke 0.5MH2SO4tharollo.

12.5 Se ke oa sebelisa lisebelisoa tse siiloeng ke nako.Se ke oa fapanyetsana li-reagents tsa lihlopha tse fapaneng, ho seng joalo e tla theola maikutlo.

12.6 Boloka lisebelisoa tsa ELISA ho 2-8 ℃, u se ke ua hatsele.Koahela lipoleiti tsa microwell tse phomolang, Qoba khanya ea letsatsi e otlolohileng nakong ea li-incubations tsohle.Ho khothalletsoa ho koahela lipoleiti tsa microtiter.

12.7 Tharollo ea substrate e lokela ho tloheloa haeba e fetola mebala.Li-reagents li ka fetoha hampe haeba boleng ba ho monya (450/630nm) ba maemo a lefela bo le ka tlase ho 0.5 (A450nm<0.5).

12.8 Karabelo ea mebala e hloka 15min ka mor'a ho eketsoa ha tharollo ea A le tharollo ea B. 'Me u ka lelefatsa nako ea ho incubation ho 20min kapa ho feta haeba' mala o le bobebe haholo hore o ka tsebahatsoa.Le ka mohla u se ke ua feta 30min, ho fapana le hoo, khutsufatsa nako ea ho incubation hantle.

12.9 Mocheso o nepahetseng oa ho arabela ke 37℃.Mocheso o phahameng kapa o tlase o tla lebisa liphetohong tsa kutloisiso le litekanyetso tsa ho monya.

13. Polokelo

Boemo ba polokelo: 2-8 ℃.

Nako ea polokelo: Likhoeli tse 12.

 


  • E fetileng:
  • E 'ngoe:

  • Ngola molaetsa wa hao mona mme o re romele wona