Kukwikwidza Enzyme Immunoassay Kit yeKuwanda Kuongorora kweTylosin
Kukwikwidza Enzyme Immunoassay Kit ye
Quantitative Analysis yeTylosin
1. Background
Tylosinmacrolide antibiotic, iyo inonyanya kushandiswa se antibacterial uye anti-mycoplasma.Strict MRLs yakasimbiswa sezvo mushonga uyu unogona kutungamirira kune zvakakomba maitiro mune mamwe mapoka.
Iyi kit chigadzirwa chitsva chakavakirwa paTEKSTO yeELISA, inokurumidza, iri nyore, yakarurama uye inonzwisisika kana ichienzaniswa neyakajairwa ongororo yezviridzwa uye inongoda maawa 1.5 mukushanda kumwe chete, inogona kuderedza zvakanyanya kukanganisa kwekushanda uye kusimba kwebasa.
2. Test Principle
Iyi kit yakavakirwa pane isina kunanga-yemakwikwi ELISA tekinoroji.Iyo microtiter matsime akaputirwa nekubatanidza antigen.Zvisaririra zveTylosin mumuenzaniso zvinokwikwidza neantigen yakavharwa pa microtiter plate yeantibody.Mushure mekuwedzera kwe enzyme yakanyorwa kuti anti-antibody, TMB substrate inoshandiswa kuratidza ruvara.Absorbance yemuenzaniso yakashata yakabatana neiyo tylosin inogara mairi, mushure mekuenzanisa neStandard Curve, yakawedzerwa ne dilution factor, tylosin residue quantity mumuenzaniso inogona kuverengwa.
3. Zvikumbiro
Iyi kit inogona kushandiswa muhuwandu uye hwemhando yekuongorora tylosin zvakasara munyama yemhuka (huku, nyama yenguruve, dhadha) nemukaka, uchi, zai, nezvimwe.
4. Kusiyana-siyana
Tylosin……………………………………………………..100%
Tilmicosin…………………………………………………<2%
5. Zvinhu Zvinodiwa
5.1 Equipments:
---- Microtiter plate spectrophotometer (450nm/630nm)
----Rotary evaporator kana nitrogen yekuomesa zviridzwa
---- homogenizer
-----Shaker
----Centrifuge
----Analytical balance (inductance: 0.01g)
---- Pipette akapedza kudzidza: 10ml
----Rubber pipette girobhu
----Volumetric flask: 10ml
----Polystyrene centrifuge tubes: 50ml
----Micropipettes: 20-200ml, 100-1000ml
250ml-multipipette
5.2 Reagents:
----Sodium hydroxide (NaOH, AR)
----Sodium bicarbonate (NaHCO3,AR)
---- Sodium carbonate (NaCO3, AR)
----Trichloroacetic acid (AR)
---- Acetonitrile (AR)
----Ethyl acetate (AR)
┅┅n-Hexane (AR)
-----Deionized mvura
6. Zvikamu zveKit
l Microtiter ndiro ine 96 matsime akaputirwa neantigen
l Yakajairwa mhinduro (5 mabhodhoro, 1ml / bhodhoro)
0ppb, 0.5ppb, 1.5ppb, 4.5ppb, 13.5ppb
l Spiking standard control: (1ml/bhodhoro)1ppm
l Enzyme conjugate 1ml……………..…..… chivharo chitsvuku
l Antibody solution 7ml……………….……kapu yegirinhi
l Solution A 7ml…………………….….……kepu yakachena
l Solution B 7ml...…………………………….. red cap
l Stop solution 7ml.…………….……….yellow cap
l 20 × yakanyanyisa kushambidza mhinduro 40ml
……………………………………..…transparent cap
l 4 × concentrated extraction solution 50ml
………………………………………………….blue cap
7. Reagents Kugadzirira:
Solution 1:0.1mol/L NaOH mhinduro
Weresa 0.4g NaOH kusvika 100ml deionized mvura uye sanganisa zvachose.
Solution 2: 1mol/L NaOH mhinduro
Weresa 4g NaOH kusvika 100ml deionized mvura uye sanganisa zvachose.
Solution 3: Carbonate buffer munyu
Solution1: 0.2M PB
Dissolve 51.6g yeNa2HPO4·12H2O, 8.7g yeNaH2PO4·2H2O nemvura ine deionized uye diluted kusvika 1000ml.
Solution2: Kubvisa mhinduro
Deresa iyo 2 × yakadzika yekubvisa mhinduro nemvura yakasvibiswa muhuwandu hwehuwandu hwe1: 1 (semuenzaniso 10ml ye2 × yekubvisa mhinduro + 10ml yemvura yakasvibiswa), iyo ichashandiswa kuenzanisa kutorwa,mhinduro iyi inogona kuchengetwa pa4 ℃ kwemwedzi 1.
Solution3: Geza mhinduro
Deredzai iyo 20 × yakadzika yekugezesa mhinduro nemvura yakasvibiswa muhuwandu hwehuwandu hwe1:19 (semuenzaniso 5ml ye20×wash solution + 95ml yemvura yakasvibiswa), iyo ichashandiswa pakusuka ndiro.Iyi mhinduro inogona kuchengetwa pa4 ℃ kwemwedzi 1.
8. Muenzaniso Kugadzirira
8.1 Chiziviso uye chenjedzo usati washanda:
(a) Ndokumbira ushandise matipi e-imwe-mukuita kuyedza, uye shandura matipi kana uchitora reagent yakasiyana.
(b) Iva nechokwadi chokuti zviridzwa zvose zvakachena.
(c) Chengetedza sampuli yetishu muchando.
(d) Sample yakagadziridzwa inofanira kushandiswa kuyedza kamwechete.
8.2 Nyama yemhuka (huku, nguruve, nezvimwewo)
---- Homogenize sampuli ne homogenizer;
----Tora 2.0±0.05g yehomogenate mu50ml polystyrene centrifuge chubhu;wedzera 2ml ye0.2M PB (mhinduro1) , zunza kunyunguduka, uye wobva wawedzera 8ml ye ethyl acetate uye zunza zvine hutsinye kwe3min;
----Centrifuge yekuparadzanisa: 3000g / ambient tembiricha / 5min.
----Kuendesa 4ml ye supernatant organic phase mu 10ml girazi chubhu, yakaoma ne 50-60 ℃ mvura yekugezera pasi pe nitrogen gasi rwizi;
----Bvisa iyo yakaoma yasara ne 1ml ye n-hexane, vortex ye 30s kuti inyunguduke, wobva wawedzera 1ml yekubvisa mhinduro (mhinduro2), vortex ye1min.centrifuge yekuparadzanisa: 3000g / ambient tembiricha / 5min
----Bvisa iyo supernatant n-hexane chikamu;tora 50μl ye substrate aqueous phase ye assay.
Dilution factor: 1
8.2 Mukaka
----Tora 100μl yesample yemukaka mbishi, sanganisa ne900μl yekubvisa mhinduro (mhinduro2), uye sanganisa zvachose.
----Tora 50μl yemhinduro yakagadzirirwa yekuongorora.
Dilution factor: 10
9. Assay maitiro
9.1 Cherechedza usati waedza
9.1.1Ita shuwa kuti ese mareagents uye mamicrowell ese ari pakamuri tembiricha (20-25 ℃).
9.1.2Dzorera mamwe mareagents ku2-8℃pakarepo mushure mekushandiswa.
9.1.3Kugeza microwells nenzira kwayo inhanho yakakosha mukuita kwekuyedza;ndicho chinhu chakakosha pakuberekazve kweiyo ELISA kuongororwa.
9.1.4 Abvisa mwenje uye uvhare ma microwell panguva yekuisa.
9.2 Matanho Ekuongorora
9.2.1 Bvisa mareagents ese kunze kwekamuri tembiricha (20-25 ℃) kweanopfuura 30min, zunza zvinyoro nyoro usati washandisa.
9.2.2 Budisa ma microwell anodiwa kunze uye wodzosera asara mubhegi rezip-lock pa2-8℃ nekukasika.
9.2.3 Mushonga wewachi yakanyungudutswa unofanirwa kudziyirwa zvakare kuti uve pakamuri tembiricha usati washandiswa.
9.2.4Nhamba:Yakaverengerwa ese ma microwell zvinzvimbo uye ese mazinga uye masampuli anofanirwa kuitiswa muduplicate.Rekodha zviyero uye masampuli nzvimbo.
9.2.5Add standard solution/sample uye antibody solution: Wedzera 50µl yeyakajairwa mhinduro ((kit yakapihwa)) kana muenzaniso wakagadzirirwa kumatsime anoenderana.Wedzera 50µl yemishonga inorwisa utachiona (kit yakapihwa)Sanganisa zvinyoro nyoro nekuzunza ndiro nemaoko uye pinza kwe30min pa 37℃ nebutiro.
9.2.6Geza: Bvisa chivharo zvinyoro uye uchenese mvura kubva mumatsime uye sukurudza ma microwell ne 250µl diluted wash solution (mhinduro3) panguva ye10s ye4-5times.Sveta mvura yasara nepepa rinonyudza (yakasara mhepo bubble inogona kubviswa nemuromo usina kushandiswa).
9.2.7Wedzera enzyme conjugate: Wedzera 100ml ye enzyme conjugate mhinduro (kit yakapihwa) kutsime rega rega, sanganisa zvinyoro nyoro uye varaire kwemaminetsi makumi matatu pa37 ℃ nekavha.Dzokorora danho rekugeza zvakare.
9.2.8ColorationWedzera 50µl yemhinduro A(kit yakapihwa) uye 50µl yemhinduro B (kit yakapihwa) kutsime rimwe nerimwe.Sanganisa zvinyoro nyoro uye incuite kwemaminetsi gumi nemashanu pa37℃ nevhavha.
9.2.9Kuyera: Wedzera 50µl yemhinduro yekumisa (kit yakapihwa) kutsime rimwe nerimwe.Sanganisa zvinyoro uye uyere kunyura pa450nm (Inokurudzirwa kuyerwa neiyo dual-wavelength ye450/630nm. Verenga mhinduro mukati me5min mushure mekuwedzera mhinduro yekumisa).
10. Migumisiro
10.1 muzana yekunyura
Izvo zvinorehwa maitiro ezviyero zvekutora zvakawanikwa kune zviyero uye sampuli dzakakamurwa nekunyura kukosha kweyero yekutanga (zero standard) uye inowedzerwa ne100%.Iyo zero chiyero inoitwa yakaenzana ne100% uye iyo yekutora tsika inotorwa muzvikamu.
B
Absorbance (%) = —— × 100%
B0
B --absorbance standard (kana sampuli)
B0 --absorbance zero standard
10.2 Standard Curve
----Kudhirowa curve yakajairwa: Tora kukosha kwekunyudza kwezviyero sey-axis, semi logarithmic yehuwandu hweiyo tylosin zviyero mhinduro (ppb) se x-axis.
----Iyo tylosin concentration yemuenzaniso wega wega (ppb), iyo inogona kuverengwa kubva pakuenzanisa curve, inowedzerwa neinowirirana Dilution factor yemuenzaniso wega wega unoteverwa, uye iyo chaiyo yekuenzanisa yemuenzaniso inowanikwa.
Tapota cherechedzai:
yakakosha software yakagadziridzwa yekuongorora data, iyo inogona kupihwa pakukumbira.
11. Sensitivity, kururama uye nemazvo
Test Sensitivity:1.5ppb
Muganho wekuona:
Nyama yemhuka………………………………………………1.5ppb Mukaka………………………………………………………..15ppb Kururama:
Zvinyama zvemhuka ……………………………………………80±15%
Mukaka…………………………………..…………..80±10%
Precision:
Kusiyanisa coefficient ye ELISA kit iri pasi pe10%.
12. Cherechedza
12.1 Izvo zvinorehwa maitiro ezviyero zvekutora zvakawanikwa kune zviyero uye sampuli zvichaderedzwa kana reagents uye masampuli asina kudzorwa kune tembiricha yekamuri (20-25 ℃).
12.2 Usabvumira mamicrowell kuti aome pakati pematanho kudzivirira kusabudirira kuberekana uye shandisa danho rinotevera nekukurumidza mushure mekutapa iyo microwells inobata.
12.3 Zunza reagent yega yega zvinyoro nyoro usati washandisa.
12.4 Chengetedza ganda rako kure nemhinduro yekumira nekuti iri 0.5MH2SO4mhinduro.
12.5 Usashandise kits dzechinyakare.Usatsinhane mareagents emabhechi akasiyana, kana zvikasadaro anodonha senitivity.
12.6 Chengetedza ELISA kits pa2-8℃, usaomesa nechando.Simbisa zororo microwell mahwendefa, Dzivisa chiedza chezuva chakatwasuka panguva dzese incubations.Kuvhara mahwendefa e microtiter kunokurudzirwa.
12.7 Substrate mhinduro inofanira kusiiwa kana ichichinja mavara.Iwo mareagents anogona kuve akashata kana iyo absorbance value (450/630nm) yezero standard iri pasi pe0.5 (A450nm<0.5).
12.8 The coloration reaction inoda 15min mushure mekuwedzera kwemhinduro A uye mhinduro B. Uye iwe unogona kurebesa nguva yeincubation kusvika ku20min kana kupfuura kana ruvara rwakanyanya kujeka kuti usarudze.Usambofa wakapfuudza 30min, pane zvinopesana, kupfupisa nguva yekuisa zvakanaka.
12.9 Iyo yakakwana yekuita tembiricha i37 ℃.Kupisa kwepamusoro kana kuderera kunotungamira kune shanduko yekunzwa uye kunyura maitiro.
13. Kuchengeta
Kuchengetedza mamiriro: 2-8 ℃.
Nguva yekuchengetedza: 12 mwedzi.
