chigadzirwa

Test Principle

Iyi kit yakavakirwa pane isina kunanga-yemakwikwi ELISA tekinoroji.Iyo microtiter matsime akaputirwa nekubatanidza antigen.Flumequinezvakasara mumucherechedzo 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 tetracycline inogara mairi, mushure mekuenzanisa neStandard Curve, yakawedzerwa ne dilution yakawanda, Flumequine yakasara yakawanda mumuenzaniso inogona kuverengwa.

Applications

Kiti iyi inogona kushandiswa mukuwongorora kwehuwandu uye hwemhando yezvakasara zveflumequine muuchi.

Cross-reactions

Flumequine ………………………………………………… 100%

Zvinhu Zvinodiwa

Equipments

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

┅┅Homogenizer kana mudumbu

┅┅Shaker

┅┅Vortex musanganisi

┅┅Centrifuge

┅┅Analytical balance (inductance: 0.01g)

┅┅Akapedza kudzidza pipette: 15ml

┅┅Rubber pipette girobhu

┅┅Polystyrene Centrifuge chubhu: 15ml, 50ml

┅┅Glasi yekuyedza chubhu:10ml

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

250ml - multipipette

Reagents

┅┅n-hexane(AR)

┅┅Methylene chloride(AR)

┅┅Acetonitrile(AR)

┅┅Deionized mvura

-----Yakanyanya hydrochloric acid(AR)

Kit Zvikamu

● Microtiter plate ine matsime 96 akaputirwa neantigen

● Standard mhinduro (6 mabhodhoro×1ml/bhodhoro)

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

● High concentration standard control:(1ml/bhodhoro)

………………………………………………………….100ppb

● Enzyme conjugate 12ml…………………………… chivharo chitsvuku

● Antibody solution 7ml …………..……..….…..green cap

● Solution A 7ml……..……………………..………..white cap

● Solution B 7ml …………….……………..………… chivharo chitsvuku

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

● 20XConcentrated wash solution 40ml

…………………………………………..…..transparent cap

●2X Extraction solution 50ml……………………… chivharo cheblue

Reagents Kugadzirira

7.1 Huchi muenzaniso

Solution 1 : 0.2 M Hydrochloric acid solution

Huremu 41.5ml Yakanyungudutswa hydrochloric acid, dilute nemvura yakasvibiswa kusvika ku500 ml.

Solution 2: Shamba mhinduro

Dhiraisai iyo yakaomeswa yekugezesa mhinduro nemvura yakasvibiswa muhuwandu hwehuwandu hwe1:19, iyo ichashandiswa kugeza mahwendefa.iyo diluted solution inogona kuchengetwa pa4 ℃ kwemwedzi 1.

Solution3: mhinduro yekubvisa

Diraisa iyo 2 × yakadzika yekubvisa mhinduro nemvura yakasvibiswa muhuwandu hwehuwandu hwe1: 1 (kana zvinoenderana nezvinodiwa), iyo ichashandiswa pakubvisa sampuli.Iyi mhinduro yakasvibiswa inogona kuchengetedzwa kwemwedzi 1 pa4 ℃.

Kugadzirira Mienzaniso

8.1 Chiziviso uye chenjedzo kune vashandisi vasati vashanda

(a) Ndokumbira ushandise matipi e-imwe-mukuita kuyedza, uye shandura matipi kana uchitora reagent yakasiyana.

(b) Ita shuwa kuti zvese zvekuedza zviridzwa zvakachena, zvikasadaro zvinozoita mhedzisiro yeassay.

8.2Muenzaniso weuchi

-----Weresa 2g±0.05g huchi sample mu50ml polystyrene centrifuge chubhu,

-----Wedzera 2ml 0.2 M Hydrochloric acid solution (Solution 1), vortex kuti uisanganise zvachose, wobva wawedzera 8ml methylene chloride, zunza ne shaker kwe5min kuti inyunguduke zvachose;

-----Centrifuge ye10 min, kanenge 3000g pane tembiricha yekamuri (20-25 ℃);

-----Bvisa chikamu chinoshamisa, tora 2 ml ye substrate organic solution kune 10 ml yegirazi chubhu. omesa pasi pasi pemvura yekugezera yeNitrogen inoyerera (50-60℃)

-----Wedzera 1 ml n-hexane,vortex yemakumi matatu, wobva wawedzera 1ml yekubvisa mhinduro(mhinduro 3), vortex zvakare kwe1min.Centrifuge ye5min, kanenge 3000g pane tembiricha yekamuri (20-25 ℃);

-----Bvisa chikamu chepamusoro, tora 50ml yekuongorora;

9. Assay maitiro

9.1 Cherechedza usati waedza

9.1.1 Iva nechokwadi chekuti mareagents nemamicrowell ese ari pakamuri tembiricha (20-25℃).

9.1.2 Dzosera mamwe mareagents ku2-8℃ pakarepo mushure mekushandiswa.

9.1.3 Kuwacha ma microwells nemazvo inhanho yakakosha muchirongwa chekuongorora;ndicho chinhu chakakosha pakudzokorora kweiyo ELISA kuongororwa.

9.1.4 Nzvenga chiedza uye vhara mamicrowell panguva yekuisa.

9.2 Matanho Ekuongorora

9.2.1 Bvisa mareagents ese kunze kwekamuri tembiricha (20-25 ℃) kweanopfuura 30min, homogenize 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:Nhamba yega yega microwell chinzvimbo uye ese zviyero uye masampuli anofanirwa kuitiswa muduplicate.Rekodha zviyero uye masampuli nzvimbo.

9.2.5Wedzera yakajairwa mhinduro/sample:Wedzera 50 µl yemhinduro yakajairwa kana sampuli yakagadzirwa kumatsime anoenderana.Wedzera 50µl antibody mhinduro.Sanganisa zvinyoro nyoro nekuzunza ndiro nemaoko uye pinza kwe30min pa25℃ nebutiro.

9.2.6Geza:Bvisa chivharo zvinyoro nyoro uye uchenese mvura kubva mumatsime uye sukurudza ma microwell ne 250µl diluted wash solution (solution 2) panguva yegumi kwe4-5 nguva.Sveta mvura yasara nepepa rinonyudza (yakasara mhepo bubble inogona kubviswa nemuromo usina kushandiswa).

9.2.8.Enzyme conjugate:Wedzera enzyme conjugate solution 100ml kune imwe neimwe tsime, Sanganisa zvinyoronyoro nekuzunza ndiro nemaoko uye incubate 30min pa25 ℃ nebutiro.Dzokorora danho rekugeza zvakare.

9.2.8Coloration:Wedzera 50µl mhinduro A uye 50µl mhinduro B patsime rega rega.Sanganisa zvinyoro nyoro nekuzunza ndiro nemaoko uye incuite kwemaminetsi gumi nemashanu pa25 ℃ nekavha (ona 12.8).

9.2.9Kuyera:Wedzera 50µl mhinduro yekumisa kune imwe neimwe tsime.Sanganisa zvinyoro nyoro nekuzunza ndiro nemaoko uye kuyera kunyura pa450nm pamhepo isina chinhu (Inokurudzirwa kuyerwa nedual-wavelength ye450/630nm. Verenga mhedzisiro mukati me5min mushure mekuwedzera kwemhinduro yekumisa. ) (Tinogona zvakare kuyera nekuona pasina kumira mhinduro muchidimbu cheELIASA chiridzwa)

Results

10.1 muzana yekunyura

Izvo zvinorehwa maitiro ezviyero zvekutora zvakawanikwa zvezviyero uye sampuli zvinopatsanurwa nehuwandu hwekutora hwekutanga (zero standard) uye inowedzerwa ne100%.Iyo zero chiyero inoitwa yakaenzana ne100% uye iyo yekutora tsika inotorwa muzvikamu.

Absorbance (%) = B/B0 ×100%

B --absorbance standard (kana sampuli)

B0 --absorbance zero standard

10.2 Standard Curve

Kudhirowa curve yakajairwa: Tora kukosha kwekunyudza kwemazinga se y-axis, semi logarithmic yekusangana kweflumequine zviyero mhinduro (ppb) se x-axis.

--- Theflumequinekusungirirwa kwemuenzaniso wega wega (ppb), iyo inogona kuverengerwa kubva pachiyero chekuenzanisa, inowedzerwa neinowirirana dilution yakawanda yemuenzaniso wega wega unoteverwa, uye iyo chaiyo yekumisikidza sampuli inowanikwa.

Kuti udzikise data yeELISA kits, yakakosha software yakagadziridzwa, iyo inogona kupihwa pakukumbira.

11. Sensitivity, kururama uye nemazvo

Test Sensitivity:0.3ppb

Huchi Sample dilution factor: 2

Detection limit

Huchi sampuli------------------------------------------------ -1ppb

Kururama

Muenzaniso weuchi --------------------------------------------- 90±20 %

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 kudzokorora uye shandisa danho rinotevera nekukurumidza mushure mekurova microwells inobata.

12.3.Homogenize reagent yega yega usati washandisa.

12.4.Chengetedza ganda rako kure nemhinduro yekumira nekuti ndiyo 2M ​​H₂SO₄mhinduro.

12.5 Usashandise kits dzechinyakare.Usatsinhanisa mareagents emabhechi akasiyana, nekuti anodonha senitivity.

12.6 Mamiriro ekuchengetedza:

Chengetedza ELISA kits pa2-8 ℃, usaomesa.Simira zororo microwell mahwendefa Dzivisa zuva rakatwasuka panguva yese yekuisa.Kuvhara mahwendefa e microtiter kunokurudzirwa.

12.7 Zviratidzo zvema reagents arikushata:

Substrate solution inofanira kusiiwa kana ikachinja mavara.

Iwo ma reagents anogona kuve akashata kana iyo yekunyudza kukosha (450/630nm) yezero standard iri pasi pe0.5 (A450nm＜0.5).

12.8 The coloration reaction inoda 15min mushure mekuwedzera Solution A uye Solution B. Uye iwe unogona kurebesa incubation nguva inotangira kubva ku20min kusvika kune yakawanda kana ruvara rwakanyanya kujeka kuti usarudze.Usambofa wakapfuudza 25min, Pane kudaro, pfupisa nguva yekuisa zvakanaka.

12.9 Iyo yakakwana yekuita tembiricha i25 ℃.Kupisa kwepamusoro kana kuderera kunotungamira kune shanduko yekunzwa uye kunyura maitiro.

13. Kuchengeta

Kuchengetedza mamiriro: 2-8 ℃.

Nguva yekuchengetedza: 12 mwedzi.