ibicuruzwa

Ihame ry'ikizamini

Iki gikoresho gishingiye ku buhanga bwa ELISA butaziguye.Amariba ya microtiter yashizwemo na antigen ihuza.Flumequineibisigara muri sample irushanwa na antigen yometse kuri plaque ya microtiter ya antibody.Nyuma yo kongeramo enzyme yanditseho anti-antibody, TMB substrate ikoreshwa mukwerekana ibara.Gukuramo icyitegererezo bifitanye isano mbi na tetracycline iba muri yo, nyuma yo kugereranya na Standard Curve, igwijwe na dilution nyinshi, ingano ya Flumequine isigaye irashobora kubarwa.

Porogaramu

Iki gikoresho kirashobora gukoreshwa mubisesengura ryinshi kandi ryujuje ubuziranenge bwa flumequine mubuki.

Kwisubiraho

Flumequine ………………… .. ……………………… 100%

Ibikoresho Birakenewe

Ibikoresho

┅┅Microtiter isahani ya ecran (450nm / 630nm)

┅┅Homogenizer cyangwa stomacher

Aker

MixVixex mixer

┅┅Centrifuge

BalanceIsesengura risesuye (inductance: 0.01g)

Pipette yarangije amashuri: 15ml

┅┅Rubber pipette bulb

┅┅Polystyrene Centrifuge tube: 15ml, 50ml

Tube Ikirahure cyo gupima ： 10ml

IcMicropipettes: 20ml-200ml, 100ml -10000ml,

250ml -mipipipette

Reagents

┅┅n-hexane (AR)

EtMethylene chloride (AR)

┅┅Acetonitrile (AR)

Water Amazi meza

----- Acide hydrochloric aside (AR)

Ibigize ibikoresho

Pl Isahani ya Microtiter ifite amariba 96 yometse kuri antigen

Solutions Ibisubizo bisanzwe (amacupa 6 × 1ml / icupa)

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

Control Igenzura ryinshi ryibanze: (1ml / icupa)

……………………………………………………… ......100ppb

● Enzyme conjugate 12ml ……………………… ... ingofero itukura

Solution Antibody solution 7ml ………… .. …… ..….… ..Icyatsi kibisi

Igisubizo A 7ml …… .. ………………… .. ……… ..umutwe wera

Umuti B 7ml ……………. ………… .. ………… umutuku

Hagarika igisubizo 7ml …………………… .. ……… umupira wumuhondo

● 20XIkibazo cyo gukaraba 40ml

……………………………………… ..… ..umutwe wuzuye

X 2X Gukuramo igisubizo 50ml ……………… ...… cap yubururu

Reagents Gutegura

7.1 Icyitegererezo cy'ubuki

Igisubizo 1: 0.2 M Hydrochloric acide

Uburemere 41.5ml Acide hydrochloric aside, uyungurura amazi ya deioni kugeza kuri ml 500.

Igisubizo 2: Karaba igisubizo

Koresha igisubizo cyogeshejwe hamwe namazi ya deionion mubipimo bya 1: 19, bizakoreshwa mugukaraba amasahani.igisubizo kivanze gishobora kubikwa kuri 4 ℃ ukwezi 1.

Igisubizo3: igisubizo cyo gukuramo

Koresha igisubizo cya 2 × cyibanze cyo kuvoma hamwe namazi ya deionion mubunini bwa 1: 1 (cyangwa biterwa nibisabwa), bizakoreshwa mugukuramo icyitegererezo.Iki gisubizo gishobora kubikwa ukwezi 1 kuri 4 ℃.

Icyitegererezo

8.1 Menyesha no kwirinda kubakoresha mbere yo gukora

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

(b) Menya neza ko ibikoresho byose byubushakashatsi bifite isuku, bitabaye ibyo bizagira ingaruka kubisubizo.

8.2Icyitegererezo cy'ubuki

----- Gupima 2g ± 0.05g icyitegererezo cyubuki muri 50ml polystyrene centrifuge,

----- Ongeramo 2ml 0.2 M Umuti wa Hydrochloric (Solution 1), vortex kugirango uyivange burundu, hanyuma ushyiremo 8ml methylene chloride, uzunguze hamwe na shaker kugirango 5min ushonga burundu;

----- Centrifuge kuminota 10, byibuze 3000g mubushyuhe bwicyumba (20-25 ℃);

----- Kuraho icyiciro ndengakamere, fata ml 2 yumuti wa substrate organic substrate kuri ml 10. ikirahure cya 10

----- Ongeramo ml 1 n-hexane, vortex kuri 30s, hanyuma ongeramo igisubizo cyo gukuramo 1ml (igisubizo 3), vortex on 1min.Centrifuge kuri 5min, byibuze 3000g mubushyuhe bwicyumba (20-25 ℃);

----- Kuraho icyiciro ndengakamere, fata 50ml yo gusuzuma;

9. Suzuma inzira

9.1 Menyesha mbere yo gusuzuma

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

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

9.1.3 Gukaraba microwells neza nintambwe yingenzi mugikorwa cyo gusuzuma;nikintu cyingenzi mugusubiramo isesengura rya ELISA.

9.1.4 Irinde urumuri kandi utwikire microwells mugihe cya incubation.

9.2 Suzuma Intambwe

9.2.1 Kuramo reagent zose mubushyuhe bwicyumba (20-25 ℃) kurenza 30min, homogenize 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:Umubare buri mwanya wa microwell hamwe nibipimo byose hamwe nicyitegererezo bigomba gukoreshwa muburyo bubiri.Andika ibipimo hamwe nicyitegererezo.

9.2.5Ongeraho igisubizo gisanzwe / icyitegererezo:Ongeramo 50 µl yumuti usanzwe cyangwa wateguye icyitegererezo kumariba ahuye.Ongeramo 50µl antibody igisubizo.Kuvanga witonze uzunguza isahani intoki hanyuma ushire 30min kuri 25 ℃ hamwe nigifuniko.

9.2.6Gukaraba:Kuramo igifuniko witonze kandi usukure amazi mumariba hanyuma woge microwells hamwe na 250µl ivanze yo gukaraba (igisubizo 2) hagati ya 10s inshuro 4-5.Kuramo amazi asigaye hamwe nimpapuro zinjiza (ikirere gisigaye kirashobora gukurwaho ninama idakoreshejwe).

9.2.8.Enzyme conjugate:Ongeramo enzyme conjugate igisubizo 100ml kuri buri riba, Vanga witonze uzunguza isahani intoki hanyuma ushiremo 30min kuri 25 ℃ hamwe nigifuniko.Ongera usubiremo intambwe yo gukaraba.

9.2.8Ibara:Ongeramo 50µl igisubizo A na 50µl igisubizo B kuri buri riba.Kuvanga witonze uzunguza isahani intoki hanyuma ushiremo iminota 15 kuri 25 ℃ hamwe nigifuniko (reba 12.8).

9.2.9Igipimo:Ongeramo 50µl guhagarika igisubizo kuri buri riba.Kuvanga witonze uzunguza isahani intoki hanyuma upime iyinjizwa kuri 450nm hejuru yumuyaga (Birasabwa gupima hamwe nuburebure bwa kabiri-bwa 450 / 630nm. Soma ibisubizo bitarenze 5min nyuma yo kongeramo igisubizo.) (Turashobora kandi gupima kubireba. nta guhagarika igisubizo mugihe gito cya ELIASA)

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.

Absorbance (%) = B / B0 × 100%

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 flumequine ibisubizo (ppb) nka x-axis.

---flumequinekwibanda kuri buri cyitegererezo (ppb), gishobora gusomwa uhereye kuri kalibrasi ya curve, igwizwa na dilution ijyanye na buri cyitegererezo cyakurikijwe, kandi intumbero nyayo yicyitegererezo iraboneka.

Kugabanya amakuru yo kugabanya ibikoresho bya ELISA, software idasanzwe yarakozwe, ishobora gutangwa kubisabwa.

11. Ibyiyumvo, ukuri kandi neza

Ikizamini：0.3ppb

Ubuki Icyitegererezo cyo kugabanya ibintu: 2

Imipaka ntarengwa

Icyitegererezo cy'ubuki ------------------------------------------------ -1ppb

Ukuri

Icyitegererezo cy'ubuki --------------------------------------------- 90 ± 20 %

Icyitonderwa

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

12. Menyesha

12.

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

12.3.Homogenize buri reagent mbere yo gukoresha.

12.4.Komeza uruhu rwawe kure yumuti uhagarara kuko ni 2M H.₂SO₄igisubizo.

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

12.6 Imiterere yo kubika:

Komeza ibikoresho bya ELISA kuri 2-8 ℃, ntugahagarike.Ikirango cyo kuruhuka microwell plaque Irinde urumuri rwizuba mugihe cyose cyububiko.Gupfuka ibyapa bya microtiter birasabwa.

12.7 Ibimenyetso kuri reagent bigenda nabi:

Substrate igisubizo igomba gutereranwa niba ihinduye amabara.

Reagent irashobora guhinduka nabi mugihe agaciro ko kwinjiza (450 / 630nm) ya zeru ya zeru kari munsi ya 0.5 (A450nm ＜ 0.5).

12.Ntuzigere urenga 25min, Ibinyuranye, gabanya igihe cyo gukora neza.

12.9 Ubushyuhe bwiza ni 25 ℃.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.