ibicuruzwa

1. 1.Amavu n'amavuko

Nitrofurans ni antibiyotike ya sintetike yagutse, ikoreshwa cyane mu musaruro w’inyamanswa kubera antibacterial na farumasi nziza.Zari zarakoreshejwe kandi mu guteza imbere ingurube, inkoko n’umusaruro w’amazi.Mu bushakashatsi bwigihe kirekire hamwe ninyamaswa zo muri laboratoire bwerekanye ko imiti yababyeyi na metabolite yabo yerekanaga kanseri na mutagenic.Ibi byatumye nitrofurans ibuza kuvura inyamaswa zikoreshwa mu gutanga ibiribwa.Imiti ya nitrofuran furaltadone, nitrofurantoin na nitrofurazone yabujijwe gukoresha mu gutunganya amatungo y’ibiribwa muri EU mu 1993, kandi byari bibujijwe gukoresha furazolidone mu 1995.

Isesengura ryibisigazwa byibiyobyabwenge bya nitrofuran bigomba gushingira ku gutahura metabolite ya tissue ihuza imiti yababyeyi ba nitrofuran.Kubera ko imiti y'ababyeyi ihindagurika cyane, kandi metabolite ya nitrofuran metabolite izagumana igihe kirekire, izo metabolite zikoreshwa nk'intego mu gutahura ihohoterwa rya nitrofurani, irimo metabolite ya Furazolidone (AOZ), metabolite ya Furaltadone (AMOZ) ), Nitrofurantoin metabolite (AHD) na metabolite ya Nitrofurazone (SEM).

AOZ-ibisigara bigenwa cyane na LC-MS cyangwa LC-MS / MS.Enzyme immunoassays, ugereranije nuburyo bwa chromatografique, yerekana ibyiza byinshi bijyanye na sensitivite, imipaka yo gutahura, ibikoresho bya tekiniki nibisabwa igihe.(Igiciro cyigihe: 45min)

1. 2.Ihame ry'ikizamini

Iki gikoresho cya ELISA cyagenewe kumenya AOZ hashingiwe ku ihame rya enzyme immunoassay itaziguye.Amariba ya microtiter yashizwemo no gufata antigen ihuza BSA.AOZ muri sample irushanwa na antigen yometse ku isahani ya microtiter ya antibody yongeyeho.Nyuma yo kongeramo enzyme conjugate, substrate ya chromogenic ikoreshwa kandi ikimenyetso gipimwa na spekitifotometero.Kwinjiza biringaniye muburyo bwa AOZ kwibanda murugero.

1. 3.Porogaramu

Iki gikoresho kirashobora gukoreshwa mubisesengura ryinshi kandi ryujuje ubuziranenge bwa AOZ murianima(imitsi, umwijima nibindi), ubuki.

1. 4.Kwisubiraho

Furazolidone metabolite (AOZ) …………………… ..100%

Furaltadone metabolite (AMOZ) …………………… <0.1%

Nitrofurantoin metabolite (AHD) …………………… <0.1%

Nitrofurazone metabolite (SEM) ………………… ...… <0.1%

Furazolidone …………………………………….… ..… 16.3%

Furaltadone …………………………………………….… <1%

Nitrofurantoin ……………………………………. …….… <1%

Nitrofurazone ………………………………………… ..… <1%

1. 5.Ibikoresho Birakenewe

5.1Ibikoresho

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

---- Rotary evaporator cyangwa ibikoresho byo kumisha azote

---- Homogenizer / stomacher

---- Shaker

---- Vortex ivanga

---- Centrifuge

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

---- Impamyabumenyi yarangije: 10ml

---- Rubber pipette

---- Flask ya Volumetric: 100ml

---- Ikirahure cy'ikirahure: 10ml

---- Umuyoboro wa polystirene centrifuge: 2ml, 50ml

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

250ul-kugwiza

5.2Reagents

---- Ethyl acetate (AR)

---- n-hexane (cyangwa n-heptane) (AR)

---- Dipotassium hydrogen fosifate trihydrate

(K₂HPO₄.3H₂O) (AR)

---- Acide yibanze ya hydrochloric (HCl, AR)

----- Methanol

---- Sodium hydroxide (NaOH, AR)

---- Amazi ya Deionised

1. 6.Ibigize ibikoresho

Isahani ya Microtiter yometse kuri antigen, amariba 96

l Ibisubizo bisanzwe (amacupa 6, 1ml / icupa)

0ppb, 0.025ppb, 0.075ppb, 0.225ppb, 0.675ppb, 2.025ppb

l Kugenzura kugenzura bisanzwe: (1ml / icupa) ....….100ppb

l Enzyme yibanze ya conjugate 1ml ...… ..umutwe

l Enzyme conjugate diluents 10ml ……… ..icyatsi kibisi

l Substrate A 7ml ……… ..............… ....… ..… ..umutwe wera

l Substrate B 7ml ……… ..............… ........….… ..umutwe

l Hagarika igisubizo 7ml cap capeti yumuhondo

l 20 × igisubizo cyogejwe cyane 40ml

……………………………………. …… capa ibonerana

l 2 × gukemura cyane ibisubizo 60ml… .umutwe wurururu

l 2-Nitrobenzaldehyde 15.1mg cap ingofero yera

1. 7.Reagents Gutegura

Igisubizo 1: inkomoko ya reagent:

Ongeramo 10ml ya methanol kumacupa ifite 2-Nitrobenzaldehyde hanyuma ushonga.(kuri concentration ya 10mM).

Igisubizo 2: 0.1MK₂HPO₄igisubizo:

Ibiro 22.8g K.₂HPO₄.3H₂O kugeza 1L y'amazi ya deionised kugirango ashonga.

Igisubizo 3: 1M HCl igisubizo

Kuramo 8.3ml Acide hydrochloric aside n'amazi ya deionised kugeza 100ml.

Igisubizo 4:1M NaOH igisubizo

Kuramo 4g NaOH n'amazi ya 100ml.

Igisubizo 5: igisubizo cyo gukuramo:

Koresha 2 × igisubizo cyibanze cyo gukuramo hamwe namazi ya deionion mubipimo bya 1: 1.Iki gisubizo kirashobora kubikwa ukwezi 1 kuri 4 ℃, kizakoreshwa mugukuramo ingero.

Igisubizo 6: gukaraba igisubizo:

Koresha igisubizo cya 20 × cyibanze cyo gukaraba hamwe namazi ya deionion mubunini bwa 1:19, bizakoreshwa mukwoza amasahani.Iki gisubizo gishobora kubikwa ukwezi 1 kuri 4 ℃.

1. 8.Icyitegererezo

8.1Menyesha no kwirinda mbere yo gukora:

a) Nyamuneka koresha inama imwe gusa mugeragezwa, hanyuma uhindure inama mugihe ukurura reagent zitandukanye.

b) Menya neza ko ibikoresho byose byubushakashatsi bifite isuku.

c) inkomoko ya reagent irashobora kubikwa kuri 2-8 ℃ amezi atatu;

d) Igisubizo cya HCl kirashobora kubikwa mubushyuhe bwicyumba amezi 3;

e) Igisubizo cya NaOH gishobora kubikwa amezi 3 mubushyuhe bwicyumba;

f) Gumana ingero zitavuwe muguhagarika (-20 ℃);

g) Ingero zakozwe zishobora kubikwa 24h kuri 2-8 ℃ mu mwijima.

8.2 Ibice byinyamanswa hamwe numwijima byintangarugero:

---- Guhuza ingero hamwe na homogenizer;

---- Uburemere 1.0 ± 0.05g ya sample ya homogenized sample sample muri 50ml polystyrene centrifuge.Ongeramo 4ml amazi ya deionised, 0.5ml 1M HCl igisubizo na 100ul derivative reagent (reba igisubizo 1).Kunyeganyeza 2min.

---- Incubate kuri 37 ℃ ijoro ryose (hafi 16h);

---- Ongeramo 5ml 0.1MK₂HPO₄ (igisubizo2), 0.4ml 1M NaOH (igisubizo4) na 5ml Ethyl acetate.Kunyeganyega cyane kuri 30;

---- Centrifuge ku bushyuhe bwicyumba (20-25 ℃) kuri 10min, byibuze 3000g;

---- Fata 2,5ml yicyiciro ndengakamere mumashanyarazi ya 10ml isukuye, yumye hamwe na 50-60 gas gaze ya azote cyangwa moteri izunguruka;

---- Kuraho ibisigara byumye hamwe na 1ml n-hexane (cyangwa n- heptane), vortex kumyaka 30, ongeramo igisubizo cya 1ml cyo gukuramo (igisubizo5), vortex 1min, vanga rwose.

---- Centrifuge ku bushyuhe bwicyumba (20-25^oC) kuri 5min, byibuze 3000g;

---- Kuraho icyiciro ndengakamere;fata 50μl yicyiciro cyamazi ya substrate kugirango usuzume.

8.4 Ubuki

---- ipima 1.0 ± 0,05g yubuki bwa homogenized sample ya 50ml polystyrene centrifuge;

---- Ongeramo 4ml amazi ya deionised, 0.5ml 1M HCl (igisubizo3) na 100μl ikomoka kuri reagent (igisubizo1);kunyeganyega rwose kuri 2min;

---- Incubate kuri 37 ℃ ijoro ryose (hafi 16h);

---- Ongeramo 5ml 0.1MK₂HPO₄ (igisubizo2), 0.4ml 1M NaOH (igisubizo4) na 5ml Ethyl acetate, kunyeganyega cyane kuri 30;

---- Centrifuge ku bushyuhe bwicyumba (20-25 ℃) kuri 10min, byibuze 3000g;

---- Fata 2,5ml yicyiciro ndengakamere mumashanyarazi ya 10ml isukuye, yumye hamwe na 50-60 gas gaze ya azote cyangwa moteri izunguruka;

---- Kuraho ibisigara byumye hamwe na 1ml n-hexane (cyangwa n- heptane), vortex kumyaka 30, ongeramo igisubizo cya 1ml cyo gukuramo (igisubizo5), vortex 1min, vanga rwose.

---- Centrifuge ku bushyuhe bwicyumba (20-25^oC) kuri 10min, byibuze 3000g;

---- Kuraho icyiciro ndengakamere;fata 50μl yicyiciro cyamazi ya substrate kugirango usuzume.

1. 9.Suzuma inzira

9.1Menyesha 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 gukoresha.

9.1.3 Gukaraba microwells neza nintambwe yingenzi mugikorwa cyo gusuzuma;nikintu cyingenzi kubyara imyororokere ya 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 hamwe nigisubizo cyakuweho bigomba gusubirwamo kugirango ubushyuhe bwicyumba mbere yo kubikoresha.

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

9.2.5Gukoresha antibody yibanze.

9.2.6Ongeraho igisubizo gisanzwe / icyitegererezo hamwe na enzyme conjugate igisubizo: ongeramo 50µl yumuti usanzwe cyangwa wateguye icyitegererezo kumariba ahuye, ongeramo 50µl enzyme conjugate igisubizo.Kuvanga witonze uzunguza isahani intoki hanyuma ushire 30min kuri 25 ℃ hamwe nigifuniko.

9.2.7Karaba: Kuraho igifuniko witonze hanyuma usukemo amazi mumariba hanyuma woge microwells hamwe na 250µl ivanze yo gukaraba (igisubizo6) hagati ya 10s inshuro 4-5.Kuramo amazi asigaye hamwe nimpapuro zinjiza (ikirere gisigaye kirashobora gukurwaho ninama idakoreshejwe).

9.2.8Ibara: Ongeramo 50µl substrate igisubizo A na 50ul substrate igisubizo B kuri buri riba.Kuvanga witonze ukunkumura isahani intoki hanyuma ugashyiramo 15min kuri 25 ℃ hamwe nigifuniko (reba 12.8).

9.2.11Igipimo: Ongeramo 50µl guhagarika igisubizo kuri buri riba.Kuvanga witonze ukanyeganyeza isahani intoki hanyuma upime iyinjizwa rya 450nm (Byasabye gupima hamwe nuburebure bwumurongo wa 450 / 630nm. Soma ibisubizo bitarenze 5min nyuma yo kongeramo igisubizo.)

10 Ibisubizo

10.1Kwinjira 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 ——ibisanzwe (cyangwa icyitegererezo)

B0 ——absorbance zeru zisanzwe

10.2Umurongo usanzwe

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

---- AOZ yibanze kuri buri sample (ppb), ishobora gusomwa uhereye kumurongo wa kalibrasi, igwizwa nimpamvu ijyanye na dilution ihuye na buri cyitegererezo cyakurikijwe, kandi intumbero nyayo yicyitegererezo iraboneka.

Nyamuneka menya:

Porogaramu idasanzwe yateguwe kugirango igabanye amakuru yose, irashobora gutangwa kubisabwa.

Impamvu……………………………………… .. …… 2

10.Ibyiyumvo, ukuri kandi neza

Ibyiyumvo: 0.025ppb

Umupaka wo gutahura:

Tissue (imitsi, umwijima) ………………………… 0.1ppb

Ubuki ------------------------------------------------- -0.1ppb

Ukuri:

Inyama zinyamaswa (imitsi numwijima) …… ... ………… 100 ± 20%

Ubuki ……………………………… .. ………… .... 100 ± 20%

Icyitonderwa:CV ya ELISA kit kiri munsi ya 10%.

11.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 Imiterere yububiko: 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 kwangirika mugihe agaciro ko kwinjiza (450 / 630nm) kerekana zeru kari munsi ya 0.5 (A450nm <0.5).

12.8 Ibara ryibara rikenera 15min nyuma yo kongeramo igisubizo cya substrate;Urashobora kwongerera igihe cya incubation kugeza 20min cyangwa irenga niba ibara ryoroheje cyane kuburyo ridashobora kugenwa., Ntuzigere urenga 25min. Kurundi ruhande, 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.

12.Imiterere yo kubika nigihe cyo kubika

Imiterere yo kubika: 2-8 ℃.

Igihe cyo kubika: amezi 12.