ibicuruzwa

Enzyme Irushanwa Immunoassay Kit kugirango Isesengura ryinshi rya Tylosine

Ibisobanuro bigufi:

Tylosine ni antibiyotike ya macrolide, ikoreshwa cyane nka antibacterial na anti-mycoplasma.MRLs ntizashizweho kuva uyu muti ushobora gutera ingaruka zikomeye mumatsinda amwe.

Iki gikoresho nigicuruzwa gishya gishingiye ku buhanga bwa ELISA, bwihuta, bworoshye, bwuzuye kandi bworoshye ugereranije nisesengura ryibikoresho bisanzwe kandi bikenera amasaha 1.5 gusa mugikorwa kimwe, birashobora kugabanya cyane amakosa yibikorwa hamwe nimbaraga zakazi.


Ibicuruzwa birambuye

Ibicuruzwa

Irushanwa Enzyme Immunoassay Kit ya

Isesengura ryinshi ryaTylosin


1. Amavu n'amavuko

Tylosinni antibiyotike ya macrolide, ikoreshwa cyane nka antibacterial na anti-mycoplasma.MRLs ntizashizweho kuva uyu muti ushobora gutera ingaruka zikomeye mumatsinda amwe.

Iki gikoresho nigicuruzwa gishya gishingiye ku buhanga bwa ELISA, bwihuta, bworoshye, bwuzuye kandi bworoshye ugereranije nisesengura ryibikoresho bisanzwe kandi bikenera amasaha 1.5 gusa mugikorwa kimwe, birashobora kugabanya cyane amakosa yibikorwa hamwe nimbaraga zakazi.

2. Ihame ry'ikizamini

Iki gikoresho gishingiye ku buhanga bwa ELISA butaziguye.Amariba ya microtiter yashizwemo na antigen ihuza.Ibisigarira bya Tylosine murugero birushanwe na antigen yometse ku isahani ya microtiter ya antibody.Nyuma yo kongeramo enzyme yanditseho anti-antibody, TMB substrate ikoreshwa mukwerekana ibara.Gukuramo icyitegererezo bifitanye isano mbi na tylosine iba muri yo, nyuma yo kugereranya na Standard Curve, igwijwe nimpamvu yo kugabanuka, ingano ya tylosine isigaye muri sample irashobora kubarwa.

3. Gusaba

Iki gikoresho gishobora gukoreshwa mubisesengura ryinshi kandi ryujuje ubuziranenge bwibisigazwa bya tylosine mubice byinyamanswa (inkoko, ingurube, inkongoro) n'amata, ubuki, amagi, nibindi.

4. Kwisubiraho

Tylosine ……………………………………………… ..100%

Tilmicosine ……………………………………………… <2%

5. Ibikoresho bisabwa

5.1 Ibikoresho:

---- Isahani ya microtiter yerekana ecran (450nm / 630nm)

---- Rotary evaporator cyangwa ibikoresho byo kumisha azote

---- homogenizer

---- Shaker

---- Centrifuge

---- Impirimbanyi zisesenguye (inductance: 0.01g)

---- Impamyabumenyi yarangije: 10ml

---- Rubber pipette

---- Flask ya Volumetric: 10ml

---- Imiyoboro ya polystirene centrifuge: 50ml

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

250ml-kugwiza

5.2 Reagents:

----Hydroxide ya Sodium (NaOH, AR)

---- Sodium bicarbonate (NaHCO3,AR)

---- Sodium karubone (NaCO3, AR)

---- Acide Trichloroacetic (AR)

---- Acetonitrile (AR)

---- Ethyl acetate (AR)

┅┅n-Hexane (AR)

---- Amazi ya Deionised

6. Ibigize ibikoresho

Isahani ya Microtiter ifite amariba 96 yometse kuri antigen

l Ibisubizo bisanzwe (amacupa 5, 1ml / icupa)

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

l Igenzura risanzwe: (1ml / icupa)1ppm

l Enzyme conjugate 1ml …………… ..… ..… ingofero itukura

l Antibody igisubizo 7ml ………………. …… cap cap

l Igisubizo A 7ml …………………….…. …… ingofero yera

l Igisubizo B 7ml ... …………………………… ..umutwe

Hagarika igisubizo 7ml. ………………. ……… .umutwe wumuhondo

l 20 × igisubizo cyogejwe cyane 40ml

………………………………… ..… ingofero iboneye

l 4 × igisubizo cyibanze cyo gukuramo 50ml

………………………………………………

7. Imyiteguro ya Reagents:

Igisubizo 1:0.1mol / L NaOH igisubizo

Gupima 0.4g NaOH kugeza 100ml amazi ya deionised hanyuma uvange rwose.

Igisubizo 2: 1mol / L NaOH igisubizo

Gupima 4g NaOH kugeza 100ml amazi ya deionised hanyuma ukavanga rwose.

Igisubizo 3: Umunyu wa karubone

Igisubizo1: 0.2M PB

Gabanya 51,6g ya Na2HPO4· 12H2O, 8.7g ya NaH2PO4· 2H2O hamwe n'amazi ya deionion hanyuma ukayungurura kugeza kuri 1000ml.

Igisubizo2: Igisubizo cyo gukuramo

Koresha igisubizo cya 2 × cyibanze cyo kuvoma hamwe namazi ya deionion mubipimo bya 1: 1 (urugero 10ml ya 2 solution igisubizo cyo gukuramo + 10ml y'amazi ya deioni), izakoreshwa mu gukuramo icyitegererezo,iki gisubizo kirashobora kubikwa kuri 4 ℃ ukwezi 1.

Igisubizo3: Karaba igisubizo

Koresha igisubizo cya 20 × cyibanze cyo gukaraba hamwe namazi ya deionione mubipimo bya 1:19 (urugero 5ml ya 20 × gukaraba igisubizo + 95ml y'amazi ya deioni), izakoreshwa mu koza amasahani.Iki gisubizo kirashobora kubikwa kuri 4 ℃ ukwezi 1.

8. Icyitegererezo

8.1 Menyesha no kwirinda mbere yo gukora:

(a) Nyamuneka koresha inama imwe murwego rwo kugerageza, hanyuma uhindure inama mugihe ushizemo reagent zitandukanye.

(b) Menya neza ko ibikoresho byose bifite isuku.

(c) Gumana icyitegererezo cya tissue mukonje.

(d) Icyitegererezo cyateguwe kigomba gukoreshwa icyarimwe icyarimwe.

8.2 Inyama zinyamaswa (inkoko, ingurube, nibindi)

---- Guhuza icyitegererezo hamwe na homogenizer;

---- Fata 2.0 ± 0.05g ya homogenate muri 50ml polystyrene centrifuge;ongeramo 2ml ya 0.2M PB (igisubizo1), kunyeganyega gushonga, hanyuma wongereho 8ml ya Ethyl acetate hanyuma unyeganyeze cyane kuri 3min;

---- Centrifuge yo gutandukana: 3000g / ubushyuhe bwibidukikije / 5min.

---- Hindura 4ml yicyiciro ndengakamere mumashanyarazi ya 10ml, yumye hamwe na 50-60 bath ubwogero bwamazi munsi yumugezi wa azote;

---- Kuramo ibisigara byumye hamwe na 1ml ya n-hexane, vortex kumyaka 30 kugirango ushonga, hanyuma wongereho 1ml yumuti wo gukuramo (igisubizo2), umuyaga kuri 1min.centrifuge yo gutandukana: 3000g / ubushyuhe bwibidukikije / 5min

---- Kuraho icyiciro ndengakamere n-hexane;fata 50μl ya substrate y'amazi yo murwego rwo gusuzuma.

 

Impamvu zikoreshwa: 1

 

8.2 Amata

---- Fata 100μl yicyitegererezo cyamata mbisi, vanga na 900μl yumuti wo gukuramo (igisubizo2), hanyuma uvange rwose.

---- Fata 50μl yumuti wateguwe kugirango usuzume.

 

Impamvu zikoreshwa: 10

 

9. Suzuma inzira

9.1 Menyesha mbere yo gusuzuma

9.1.1Menya neza ko reagent zose na microwells byose biri mubushyuhe bwicyumba (20-25 ℃).

9.1.2Subiza reagent zose zisigaye kuri 2-8ako kanya nyuma yo gukoreshwa.

9.1.3Gukaraba microwells neza nintambwe yingenzi mugikorwa cyo gusuzuma;nikintu cyingenzi kubyara imyororokere ya ELISA.

9.1.4 A.gusiba urumuri hanyuma utwikire microwells mugihe cya incubation.

9.2 Suzuma Intambwe

9.2.1 Kuramo reagent zose mubushyuhe bwicyumba (20-25 ℃) kurenza 30min, uzunguze buhoro mbere yo kuyikoresha.

9.2.2 Shakisha microwells ikenewe hanyuma usubize ahasigaye mumifuka ya zip-lock kuri 2-8 ℃ ako kanya.

9.2.3 Igisubizo cyo gukaraba kigomba gusubirwamo kugirango ubushyuhe bwicyumba mbere yo kugikoresha.

9.2.4Umubare:Kubara buri mwanya wa microwell hamwe nibipimo byose hamwe nicyitegererezo bigomba gukoreshwa muburyo bubiri.Andika ibipimo hamwe nicyitegererezo.

9.2.5Add igisubizo gisanzwe / icyitegererezo hamwe na antibody igisubizo: Ongeraho 50µl yumuti usanzwe ((ibikoresho byatanzwe)) cyangwa yateguye icyitegererezo kumariba ahuye.Ongeramo 50µl yumuti wa antibody (ibikoresho byatanzwe).Kuvanga witonze uzunguza isahani intoki hanyuma ushire 30min kuri 37 ℃ hamwe nigifuniko.

9.2.6Karaba: Kuraho igifuniko witonze kandi usukure amazi mumariba hanyuma woze microwells hamwe na 250µl ivanze yo gukaraba (igisubizo3) hagati ya 10s kuri 4-5times.Kuramo amazi asigaye hamwe nimpapuro zinjiza (ikirere gisigaye kirashobora gukurwaho ninama idakoreshejwe).

9.2.7Ongeramo enzyme conjugate: Ongeramo 100ml ya enzyme conjugate igisubizo (ibikoresho byatanzwe) kuri buri riba, vanga witonze kandi ushyire 30min kuri 37 ℃ hamwe nigifuniko.Ongera usubiremo intambwe yo gukaraba.

9.2.8Ibara: Ongeramo 50µl yumuti A (ibikoresho byatanzwe) na 50µl yumuti B (ibikoresho byatanzwe) kuri buri riba.Kuvanga witonze hanyuma ushire 15min kuri 37 ℃ hamwe nigifuniko.

9.2.9Igipimo: Ongeramo 50µl yo guhagarika igisubizo (ibikoresho byatanzwe) kuri buri riba.Kuvanga witonze hanyuma upime ibyinjira kuri 450nm (Birasabwa gupima hamwe nuburebure bwa kabiri-450 / 630nm. Soma ibisubizo bitarenze 5min nyuma yo kongeramo igisubizo gihagarara).

10. Ibisubizo

10.1 Kwinjira kw'ijana

Indangagaciro zingana zo kwinjiza agaciro zabonetse kubipimo hamwe nicyitegererezo bigabanijwe nigiciro cyo kwinjiza cyambere (zeru zeru) kandi cyikubye 100%.Ibipimo bya zeru rero bikozwe bingana na 100% kandi indangagaciro zo kwinjiza zavuzwe ku ijanisha.

B

Absorbance (%) = —— × 100%

B0

B ——ibisanzwe (cyangwa icyitegererezo)

B0 ——absorbance zeru zisanzwe

10.2

---- Gushushanya umurongo usanzwe: Fata agaciro ko kwinjiza ibipimo nka y-axis, igice cya logarithmic yo kwibanda kumurongo wa tylosine (ppb) nka x-axis.

---- Kwibanda kwa tylosine ya buri sample (ppb), ishobora gusomwa uhereye kumurongo wa kalibrasi, igwizwa ningingo ijyanye na Dilution ihuye na buri cyitegererezo cyakurikijwe, kandi habonetse kwibumbira hamwe kwicyitegererezo.

Nyamuneka menya:

porogaramu idasanzwe yateguwe kugirango isesengurwe ryamakuru, irashobora gutangwa kubisabwa.

11. Ibyiyumvo, ukuri kandi neza

Kwiyumvisha Ikizamini:1.5ppb

Umupaka wo gutahura:

Inyama zinyamaswa …………………………. …………… 1.5ppb Amata …………………………………………… .....… ..15ppb Ukuri:

Inyama zinyamaswa ………………………… ... ………… 80 ± 15%

Amata ………………………………… .. ………. …… 80 ± 10%

Icyitonderwa:

Coefficient yo gutandukanya ibikoresho bya ELISA iri munsi ya 10%.

12. Menyesha

12.

12.2 Ntukemere ko microwells yumisha hagati yintambwe kugirango wirinde kubyara bitagenze neza kandi ukore intambwe ikurikira ako kanya nyuma yo gukanda kuri microwells.

12.3 Shyira buri reagent witonze mbere yo kuyikoresha.

12.4 Komeza uruhu rwawe kure yumuti uhagarara kuko ni 0.5MH2SO4igisubizo.

12.5 Ntukoreshe ibikoresho bitajyanye n'igihe.Ntugahindure reagent yibice bitandukanye, bitabaye ibyo bizagabanya sensibilité.

12.6 Gumana ibikoresho bya ELISA kuri 2-8 ℃, ntugahagarike.Funga ikiruhuko cya microwell plaque, Irinde urumuri rwizuba rugororotse mugihe cyose.Gupfuka ibyapa bya microtiter birasabwa.

12.7 Substrate igisubizo igomba gutereranwa niba ihinduye amabara.Reagent irashobora guhinduka nabi mugihe agaciro ko kwinjiza (450 / 630nm) ya zeru ya zero iri munsi ya 0.5 (A450nm <0.5).

12.Ntuzigere urenga 30min, kurundi ruhande, gabanya igihe cyo gukora neza.

12.9 Ubushyuhe bwiza ni 37 ℃.Ubushyuhe bwo hejuru cyangwa hasi buzaganisha ku mpinduka zo kwiyumvisha ibintu no kwinjiza agaciro.

13. Ububiko

Imiterere yo kubika: 2-8 ℃.

Igihe cyo kubika: amezi 12.

 


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