Umhlahlandlela Wokuthuthukiswa Kwe-Assay Assortment

I-Lateral Flow Assay iyithuluzi elisetshenziselwa ukuhlolwa okusheshayo ekhaya kanye nokuhlolwa kwemitholampilo. Yizinto ezithembekile nezinembile kakhulu ezisetshenziswa cishe kuwo wonke amazwe athuthukile nasathuthuka. Futhi, i- lateral flow Assay Development Guide inezinyathelo ezingaba yi-10 ezinquma inqubo yokuthuthukiswa kwe-Lateral Flow Assay. Ngokuvamile, i-Lateral Flow Assays ine-sample pad, i-conjugate pad, i-nitrocellulose membrane, i-labels adsorbent pad, kanye ne-buffer. Kodwa-ke, kungaba nezinguquko ezithile kumalebula njengoba kunezinhlobo ezahlukene zamalebula ezisetshenziswa ekuhlolweni kwe-lateral flow.

Ukuze uqale umhlahlandlela wokuthuthukiswa kwesilingo sokugeleza kwe-lateral , kubalulekile ukucabangela izinyathelo ezimbalwa ngaphambi kokuqala inqubo. Okokuqala, kubalulekile ukuhlaziya ifomethi okufanele ikhethwe yesilingo sokugeleza kwe-lateral ukuthi kungaba ukuncintisana noma ifomethi ye-sandwich. Ngokuyisisekelo, umhlahlandlela wokuthuthukiswa kwe-Lateral flow Assay uncike enkambisweni yokuthuthukiswa kwesilingo. Isinyathelo sokuqala esibucayi nesibaluleke kakhulu ukuhlonza isilingo sokulawula esizosiza ekuboneni i-target analyte kusampula. Lo mhlahlandlela wokuthuthukiswa uzonikeza inqubo yokuthuthukiswa kwe-LFA ejwayelekile ngoba izilingo ezahlukene nazo zinenqubo ehlukile.

Isinyathelo sokuqala somhlahlandlela wokuthuthukiswa ukukhethwa kwama-nanoparticles. Ukukhethwa kwama-nanoparticles kuyahlukahluka ngobukhulu kanye nesimo. Kunezinhlobo ezahlukene zosayizi ezitholakalayo zokuhlolwa kokugeleza kwe-lateral. Kukhona futhi nama-nanoparticles egolide angu-40nm ane-carbon noma i-citrate surface engasetshenziswa kakhulu enkambisweni yokumunca amaprotheni. Amanye ama-nanoparticles angakhethwa ekuthuthukisweni kwe-LFA afaka i-carboxyl gold, ama-nanoshell egolide e-carboxyl, njll. ngaphandle kwalokho kunezinye izinhlayiya ezifana nama-cellulose beads, i-polystyrene edaywe, kanye nama-fluorescent probes. Iphuzu elihle lokukhethwa kwama-nanoparticles egolide e-carboxyl ukuthi enkambisweni yokumunca kudingeka ama-antibodies ambalwa kakhulu futhi ngenxa yala ma-nanoparticles kwakheka isibopho esiyisibopho se-amide esizinzile nesingenakuguqulwa. Kodwa-ke, kumelwe kukhunjulwe ukuthi amandla avelayo atholakala phakathi kwama-nanoparticles nama-antibodies abangelwa yindawo yawo yokuxhuma.

Isinyathelo sesibili ukukhethwa kwe-Antibody. Ukukhethwa kwama-antibody kuyisinyathelo esibucayi njengoba ukusebenza kwama-LFA kuncike ekubambeni kwama-antibodies, kinetics, nezinye izakhiwo ze-steric. Futhi, ukubopha kwama-antibodies kubalulekile ekutholeni imiphumela. Esinye isici esibalulekile ngenkathi kukhethwa ama-antibody yizinhlobo ezimbili zazo. Izinhlobo ezimbili zama-antibody ziyi-monoclonal ne-polyclonal. Ama-antibody e-monoclonal kulula ukukhiqizwa kanti ama-antibody e-polyclonal anokuzwela okuphezulu uma kuqhathaniswa ne-monoclonal. Esigabeni senqubo yokukhetha ama-antibody, kunezinyathelo ezimbili ezengeziwe okungukuthi ukuhlanzwa kwe-antibody, kanye ne-antibody line control. Ekukhetheni ama-antibody, isiqu esiphakanyisiwe ukuhlola ama-antibody ngesizathu sokuthi kufanele ahlolwe ngaphambi kwesikhathi. Ama-antibody angahlolwa futhi ahlolwe ngokwakha inguqulo yokuqala neyokuqala ye-lateral flow assay engasebenza njengedivayisi yesampula yokuhlola. Ukusebenza kwama-antibody kuhlukile ngohlobo oluhlukile lwama-LFA. Isibonelo, ku-ELISA i-kinetics ayibalulekanga kangako. Futhi uma ukhuluma nge-lateral flow assay, kunezimo ezithile ama-antibodies okufanele akwazi ukuzenza. Okokuqala, ama-antibodies kufanele asebenze noma ngabe ehlanganiswe nama-nanoparticles futhi ubuqotho bawo besakhiwo akufanele bulimale nganoma iyiphi indlela. Futhi, kufanele babe nekhono lokunikeza ukuphendula kwabo ngokushesha lapho isampuli iphinda ivuselelwa kabusha ngesampula. Isikhathi sokufukamela kwezinye izinhlobo zokuHlolwa isikhathi eside uma siqhathaniswa ne-Lateral Flow Assay lapho ukubophela kulayini wokuhlola kufanele kwenziwe khona phakathi nesikhathi semizuzwana embalwa.

Inqubo yesithathu kule nqubo ulwelwesi lwe-nitrocellulose futhi iyingxenye ebaluleke kakhulu ye-LFA njengoba inezintambo zokuhlola nokulawula. Ulwelwesi lwe-nitrocellulose lunama-reagents asebenza ukukhiqiza imiphumela. Itholakala ngosayizi abahlukene, ama-porosities, kanye nemiklamo. Empeleni, ulwelwesi lwe-nitrocellulose luncike esikhathini sokugeleza kwe-capillary. Ngesikhathi sokugeleza kwe-capillary, kusho ukuthi isikhathi esidingekayo sesampula ukugeleza size sifike ku-4cm. Kulesi sinyathelo, kunesinye isinyathelo esibizwa ngokuthi yi-membrane stripping. I-buffer lapho kuneprotheyini ethile enemigqa ingaba nomthelela omkhulu emiphumeleni yokugcina yanoma iyiphi i-lateral flow assay. Yize amaprotheni amaningi engahlakazwa ku-1x PBS, amanye abucayi kakhulu kwi-pH, ukumuncwa kasawoti, kanye nokuvela kwesixazululi i-trehalose esingaqanjwa ngaso. Ukwehliswa ulwelwesi esinye isinyathelo esibalulekile esiza nalesi sinyathelo. Kulesi sinyathelo, ukwakheka kokuhlolwa kwe-lateral flow assay kungumugqa wokuhlola nokulawula imigqa ye-antibody ekhona kulwelwesi lwe-nitrocellulose. Okucatshangwayo okubalulekile kulesi sinyathelo kukweluketshezi lwe-nitrocellulose lapho kuthiwa alugcini ulwelwesi lwe-nitrocellulose endaweni lapho kungekho mswakama kunalokho lonke lomisiwe. Ngokwentuthuko ye-lateral flow assay, izinga lokushisa le-membrane ye-nitrocellulose kufanele libe nomswakama. Njengoba kunezinhlobo ezahlukahlukene zokuhlolwa, okungukuthi ifomethi yokuncintisana nefomethi yesangweji nayo ebaluleke kakhulu. Ukuhlungwa kwama-antibody kuyahluka kuwo womabili amafomethi wefomethi yesangweji umhlanganiso onconywayo we-antibody yi-1mg / mL yama-antibody womugqa wokuhlola nokulawula kuphela futhi lo mkhawulo ungafika ku-0.5 kuye ku-2 mg / mL. Esigabeni se-nitrocellulose ulwelwesi kanye nomhlahlandlela wokuthuthuka kokugeleza kokuhamba, esinye isinyathelo esifakiwe yisinyathelo sokuvimba ulwelwesi. Ukwenza lesi sinyathelo kunezinzuzo eziningi njengokugeleza okuthuthukisiwe koketshezi, ukuzinza kwemichilo yokuhlola, nokuvimba ukubopha okungacacisiwe ngaleyo ndlela kuveza imiphumela eyiqiniso. Kakhulu kolwelwesi lwe-nitrocellulose ku-lateral flow assay, kunesixazululo sebhizinisi esingezelwa kulokho okwenza ulwelwesi lube yi-hydrophilic.

Isinyathelo esilandelayo i-conjugate ne-conjugate pad. Ekwenzeni i-lateral flow assay kubalulekile ukuthi uthuthukise imingcele yokuhlanganisa. Lapho i-conjugate ilungiselelwe i-lateral flow assay iyakhishwa futhi yomiswe kuphedi yokuhlangana. Ekwakheni ukuhlangana okuzinzile kwama-antibodies kungaba nezinyathelo eziningana. Izinyathelo ezithintekayo ekwakheni ukusebenza ngokubumbana nokuqinisiwe kokuhlangana. Kulesi sinyathelo, ukuzinza kokuhlangana kuyisinyathelo esibaluleke kakhulu ngakho-ke, ukuzinza kwe-colloidal kufanele kuthuthukiswe ngenkathi kuklanywa i-LFA. uzinzo lwama-colloidal lubaluleke kakhulu ekuhlolweni kwe-lateral flow ngakho-ke ngenkathi kuklanywa nokwenza ngcono i-LFA lokhu kugcinwa kucatshangelwa. Ama-nanoparticles egolide adlala indima ebalulekile ekuhlanganisweni kwe-LFA anezici ezibonakalayo ezihlukile ngosizo lwawo amunca futhi asabalalise ukukhanya ngokunemba nangokusebenza. Ukusebenzisana okunamandla phakathi kwama-nanoparticles nokukhanya kwenzeka ngenxa yama-elektroni wokuqhuba akhona ebusweni bensimbi okuholela ekusongekeni okuhlangene lapho ama-electron ejabula ngokukhanya ngemvamisa efanayo neyilinganayo. Ngemuva kokuthi kube nokuhlangana okuyimpumelelo kunemiphumela ethile kuzimpawu ezibonakalayo njengoba ushintsho lubonwa enkombeni ye-refractive engalandelwa ngokuqhubekayo ku-UV-vis ngokuhlukaniswa kolayini obomvu. Futhi ekwelapheni i-conjugation pad, kungaba nezindlela ezahlukahlukene. Ezinye zazo ukucwilisa, noma ukufafaza ngokufanayo ngosizo lomshini wokuhambisa okuzenzakalelayo.

Ngemva kwalokhu, inqubo yokukhetha iphedi yesampula iyenzeka. Lena yingxenye yokuqala yokuhlolwa kokugeleza kwe-lateral ngakho-ke kubalulekile ukuhlola izinto zephedi yesampula kanye nokwelashwa kwangaphambili kwephedi yesampula ukuze kutholakale ukugeleza kwayo nokusebenza kwayo. Amaphedi esampula asetshenziselwa ukuhlolwa kokugeleza kwe-lateral ahlukene njenge-fiber, ukotini, okwenziwe, njll. Kuye kwaphawulwa nokuthi amanye amasampula afana nomchamo, igazi, njll. angahlukahluka ngenxa yezinye izici eziningi ezifaka phakathi izici zebhayoloji, isikhathi sokudla, isiphuzo esithathwe ngaphambi kokuqoqwa kwesampula kudivayisi yokuhlola ukugeleza kwe-lateral, nezinye eziningi. Ukuze welaphe iphedi yesampula futhi uyilungiselele, i-buffer elungiselelwe ingasetshenziswa enezinzuzo eziningi. Ngenkathi uthuthukisa ukuhlolwa kokugeleza kwe-lateral, kanye nokukhetha iphedi yesampula, i-buffer eyenziwe nge-ootmised ingasiza ekuthuthukiseni ukusebenza kwe-assay ngokunciphisa ukuguquguquka kwesampula okuhlanganisa amaprotheni, i-pH, i-viscosity, ukuhlushwa kukasawoti, njll. Kungathuthukisa futhi ukugeleza kanye nokuvumelana kwe-assay ngenkathi ithuthukiswa. Ama-buffer okwelapha abonakale ewusizo kakhulu ekugcineni ukugeleza nokuvumelana, enza i-pH ibe ngokwejwayelekile, asebenza njengezinto ezivimbayo ngokuhlanganiswa kwamaprotheni, usawoti, ama-surfactants ngokuhlushwa okufanele ngakunye. Incazelo ebaluleke kakhulu yephedi yesampula okufanele ihlolwe amandla okumunca ngoba lokho kusho ngevolumu yesampula yokuhlolwa.

Ngemuva kokuthi kukhethwe iphedi yesampula ukhetho lwe-wick pad luye olulandelayo. Iphedi yokukhotha iyasiza kwinqubo yokukhangisa yama-reagents engathathwanga yilayini ngamunye okusho ulayini wokuhlola nolayini bokulawula. Iphedi yokukhotha futhi imisa ukugeleza okuya emuva koketshezi olusetshenziswa njengesampula. Kuphakanyiswa ukuthi ngenkathi kukhethwa okokusebenza kwephedi yokukhotha kufanele kubhekwe okokugcina okokugcina okokusebenza kunoma ikuphi ukungahambisani okungaholela ezingqinambeni futhi mhlawumbe kube nemiphumela engamanga. Ngaphandle kokujwayelekile, okokujiya noma iphedi yokukhotha ayingcono kunento encanyana. Izinto ezincanyana zingcono ekwenzeni kangcono futhi imvamisa inezinto ezinamandla amakhulu kwesinye isikhathi. Kepha yonke into kufanele ihlolwe izici zayo kanye nokufaneleka kwayo ekuthuthukiseni i-lateral flow assay. Ngokukhetha usayizi nempahla yephedi yokuxubha kuyadingeka ukuyigcina ihambisane nokuthi umthamo wokumunca kufanele ube ngaphezulu futhi ivolumu yebhafa kufanele isebenze.

Ekuhlolweni kokugeleza kwe-lateral, umhlangano wokuhlolwa komugqa uyisinyathelo somhlahlandlela wawo wokuthuthuka. Emhlanganweni wokuhlolwa kwemichilo, ama-LFA aqoqiwe ngokususelwa ekukhiqizeni okukhulu noma ekukhiqizeni abancane. Kwezinye izinhlobo zokuhlolwa kwe-lateral flow, ukufundwa kokulinganiswa kungenzeka kungabi yimfuneko kepha ifomethi yediploma izodingeka ukwenza umhlangano wokuhlolwa kwe-strip ku-LFA. Kukhona ikhasethi elinokuhlolwa kwemichilo ngaphakathi kungenye yezinyathelo ezibaluleke kakhulu futhi ezibaluleke kakhulu ukufeza isivivinyo esizalwayo futhi esinokwethenjelwa. Ukucaciswa okubili okukhulunywe ngakho ngaphambili kubaluleke kakhulu ezivivinyweni eziningi, ikakhulukazi. Umsebenzi omkhulu wekhasethi ukuhlinzeka ingcindezi edingekayo koketshezi. Lokhu kwenzelwa ukuqinisekisa ukuthi kukhona ukugeleza okuhle futhi uketshezi nalo ludlula emhlanganweni lapho kugeleza okufanayo. Ikhasethi libuye liqiniseke ukuthi uketshezi aluhlali luyimiphumela emaphethelweni futhi luvele lugeleze kumaphakethe.

Ngemuva kwenhlangano yokuhlolwa komugqa, isinyathelo esilandelayo sisebenzisa isilingo. Kunezindlela ezahlukahlukene zokusebenzisa i-assay efana ne-assay yesifunda ku-liquid conjugate, i-assays ephelele ye-conjugate eyomile, ne-buffer esebenzayo. Ukuhlaziywa kwesiqeshana, kufanele kwaziwe ukuthi ngabe isivivinyo siyikhwalithi noma siyinqwaba futhi kufanele kukhethwe indlela efanele yokuhlaziya. Ukuhlaziya i-assay, kungenziwa ngeso lenyama lapho kuzoba khona imiphumela ye-yebo / cha noma ngokuthwebula umbono womugqa wokuhlola, isithwebuli se-flatbed noma ikhamera enokukhanya okulawulwayo kungasetshenziswa. Isinyathelo sokugcina ukwenziwa kwesilingo lapho kukhethwa khona i-antibody pair elungile, ulwelwesi lwe-nitrocellulose, i-wick pad, i-adsorbent pad, nazo zonke ezinye izingxenye ezifakiwe.

Isiphetho:

Umhlahlandlela wokuthuthukiswa kwe-Lateral Flow Assay unezinyathelo eziyisishiyagalolunye kuya kweziyishumi ezinikeziwe okumele zilandelwe ngokucophelela ukuze kudalwe i-lateral flow assay.

Okubhekwayo

https://cdn.shopify.com/s/files/1/0257/8237/files/BioReady_Lateral_Flow_Handbook.pdf