sehlahisoa

Teko Molao-motheo

Setsi sena se ipapisitse le theknoloji e sa tobang ea tlholisano ea ELISA.Liliba tsa microtiter li koahetsoe ka li-antigen tse kopanyang.Flumequinemasalla a 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 tetracycline e lulang ho eona, ka mor'a ho bapisa le Standard Curve, e atolositsoeng ke dilution multiple, Flumequine masala a mangata ka sampuli e ka baloa.

Lisebelisoa

Setha sena se ka sebelisoa tlhahlobong ea bongata le ea boleng ba masalla a flumequine maheng a linotsi.

Liphetoho tse fapaneng

Flumequine ………………………………………………… 100%

Lintho Tse Hlokehang

Thepa

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

┅┅Homogenizer kapa stomacher

┅┅Shaker

┅┅ Motsoako oa Vortex

┅┅Centrifuge

┅┅Ho leka-lekana ha tlhahlobo (inductance: 0.01g)

┅┅Pipette e fumaneng mangolo: 15ml

┅┅Lebone la pipette ea rabara

┅┅Polystyrene Centrifuge tube: 15ml, 50ml

┅┅Tube ea tlhahlobo ea khalase: 10ml

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

250ml - multipipette

Li-reagents

┅┅n-hexane(AR)

┅┅Methylene chloride(AR)

┅┅Acetonitrile(AR)

┅┅Metsi a senyehileng

-----Hydrochloric acid (AR)

Likarolo tsa Kit

● Microtiter plate e nang le liliba tse 96 tse tlositsoeng ka antigen

● Litharollo tse tloaelehileng (6 libotlolo×1ml/botlolo)

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

● Taolo ea maemo a holimo:(1ml/botlolo)

…………………………………………………………….100ppb

● Enzyme conjugate 12ml……………………………………

● Antibody solution 7ml …………..……..….…..kepisi e tala

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

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

● Stop solution 7ml ……………………..………kepisi e mosehla

● 20XConcentrated ho hlatsoa tharollo 40ml

……………………………………………..…..transparent cap

●2X Extraction solution 50ml……………………

Ho lokisoa ha li-reagents

7.1 Mohlala oa mahe a linotsi

Tharollo 1 : 0.2 M Hydrochloric acid solution

Weight 41.5ml Concentrated hydrochloric acid , hlapolla ka metsi a deionized ho 500 ml.

Tharollo ea 2: Hlatsoa tharollo

Hlatsoa tharollo ea ho hlatsoa e teteaneng ka metsi a hloekisitsoeng ka tekanyo ea 1:19, e tla sebelisoa ho hlatsoa lipoleiti.tharollo e hlapolotsoeng e ka bolokoa ho 4 ℃ bakeng sa khoeli e le 'ngoe.

Tharollo3: tharollo ea ho ntša

Hlakola tharollo ea 2 × concentrated ka metsi a ntšitsoeng ka tekanyo ea 1: 1 (kapa ho itšetlehile ka tlhokahalo), e tla sebelisoa bakeng sa ho ntša sampole.Tharollo ena e hlapollotsoeng e ka bolokoa khoeli e le 1 ho 4℃.

Litokisetso tsa Mehlala

8.1 Tsebiso le litemoso bakeng sa basebelisi 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 tsa liteko li hloekile, ho seng joalo li tla hlahisa sephetho sa tlhahlobo.

8.2Mohlala oa mahe a linotsi

-----Bea 2g±0.05g sampole ea mahe a linotsi ka har'a 50ml polystyrene centrifuge tube,

-----Eketsa 2ml 0.2 M Hydrochloric acid solution (Tharollo 1), vortex ho e kopanya ka ho feletseng, ebe o eketsa 8ml methylene chloride, sisinya ka shaker bakeng sa 5min ho qhibiliha ka ho feletseng;

-----Centrifuge bakeng sa 10 min, bonyane 3000g mocheso oa kamore (20-25℃);

-----Tlosa mokhahlelo o phahametseng tlhaho, nka 2 ml ea motsoako oa motsoako oa motsoako ho tube ea khalase ea 10 ml.

-----Eketsa 1 ml n-hexane,vortex bakeng sa 30s, ebe u eketsa 1ml extract solution(tharollo 3) ,vortex hape bakeng sa 1min.Centrifuge bakeng sa 5min, bonyane 3000g mocheso oa kamore (20-25 ℃);

-----Tlosa mohato o moholo, nka 50ml bakeng sa tlhahlobo;

9. Mokhoa oa ho hlahloba

9.1 Hlokomela pele ho tlhahlobo

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

9.1.2 Khutlisetsa li-reagents tsohle ho 2-8℃ hang ka mor'a hore li sebelisoe.

9.1.3 Ho hlatsoa li-microwell ka nepo ke mohato oa bohlokoa tšebetsong ea tlhahlobo;ke ntlha ea bohlokoa ho ipheta-pheta ha tlhahlobo ea ELISA.

9.1.4 Qoba lebone 'me u koahele li-microwell nakong ea ho incubation.

9.2 Mehato ea Tekolo

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

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:Nomoha boemo bo bong le bo bong ba microwell mme litekanyetso tsohle le lisampole li lokela ho tsamaisoa ka likopi.Ngola maemo le lisampole maemo.

9.2.5Kenya tharollo e tloaelehileng / mohlala:Kenya 50 µl ea tharollo e tloaelehileng kapa sampole e lokisitsoeng lilibeng tse tsamaellanang.Kenya 50µl antibody tharollo.Kopanya ka bonolo ka ho sisinya poleiti ka letsoho 'me u e kenye metsotso e 30 ho 25 ℃ ka sekoahelo.

9.2.6Hlatsoa:Tlosa sekoahelo ka bonolo 'me u hloekise mokelikeli ka ntle ho liliba' me u hlatsoe li-microwells ka 250µl e hlatsoitsoeng ea ho hlatsoa tharollo (tharollo 2) ka nako ea 10s bakeng sa linako tse 4-5.Monya metsi a setseng ka pampiri e monyang (bubble e setseng ea moea e ka felisoa ka ntlha e sa sebelisoeng).

9.2.8.Khokahano ea enzyme:Eketsa tharollo ea enzyme ea 100ml selibeng se seng le se seng, Kopanya ka bonolo ka ho sisinya poleiti ka letsoho ebe u e kenya ka metsotso e 30 ho 25 ℃ ka sekoahelo.Pheta mohato oa ho hlatsoa hape.

9.2.8Mebala:Kenya tharollo ea 50µl A le 50µl tharollo ea B selibeng ka seng.Kopanya ka bonolo ka ho sisinya poleiti ka letsoho 'me u e kenye metsotso e 15 ho 25 ℃ ka sekoahelo (sheba 12.8).

9.2.9Lekanya:Kenya 50µl tharollo ea ho emisa selibeng ka seng.Kopanya ka bonolo ka ho sisinya poleiti ka letsoho 'me u lekanye monyetla ho 450nm khahlano le moea o se nang letho (Ho khothaletsoa tekanyo e nang le maqhubu a mabeli a 450/630nm. Bala sephetho ka metsotso e 5 ka mor'a ho eketsa tharollo ea ho emisa. ) (Re ka boela ra lekanya ka pono. ntle le tharollo ea ho emisa ka bokhuts'oane ba sesebelisoa sa ELIASA)

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 (tekanyetso ea zero) '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.

Absorbance (%) = B/B0 ×100%

B - - absorbance standard (kapa sampole)

B0 -- absorbance zero standard

10.2 Mokokotlo o Tloaelehileng

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

--- Theflumequineconcentration ea sampole e 'ngoe le e 'ngoe (ppb), e ka baloang ho tloha mothapong oa calibration, e atisoa ke dilution multiple multiple ea sampole ka 'ngoe e lateloa, 'me mahloriso ea sebele ea sampole e fumanoa.

Bakeng sa phokotso ea data ea lisebelisoa tsa ELISA, ho entsoe software e khethehileng, e ka fanoang ka kopo.

11. Kutloelo-bohloko, ho nepahala le ho nepahala

Boikutlo ba Teko:0.3ppb

Honey Sample dilution factor: 2

Moeli oa ho lemoha

Mohlala oa mahe a linotsi------------------------------------------------ -1ppb

Ho nepahala

Mohlala oa mahe a linotsi --------------------------------------------- 90±20 %

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 hore li ome lipakeng tsa mehato ho qoba ho pheta-pheta ho sa atleheng le ho sebetsa mohato o latelang hang ka mor'a ho tlanya sets'oants'o sa li-microwell.

12.3.Homogenize reagent e 'ngoe le e' ngoe pele u e sebelisa.

12.4.Boloka letlalo la hau hole le tharollo ea ho emisa hobane ke 2M H₂SO₄tharollo.

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

12.6 Boemo ba polokelo:

Boloka lisebelisoa tsa ELISA ho 2-8 ℃, u se ke oa hoama.Koahela lipoleiti tsa microwell tse phomolang Qoba khanya ea letsatsi e otlolohileng nakong eohle ea ho incubation.Ho khothalletsoa ho koahela lipoleiti tsa microtiter.

12.7 Lipontšo tsa hore li-reagents ha li tsamaee hantle:

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 eketsa Tharollo A le Tharollo B. 'Me u ka lelefatsa nako ea ho incubation ho tloha ho 20min ho ea ho feta haeba' mala o le bobebe haholo hore o ka tsejoa.Le ka mohla u se ke ua feta 25min, Ho fapana le hoo, khutsufatsa nako ea ho incubation hantle.

12.9 Mocheso o nepahetseng oa ho arabela ke 25℃.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.