Isikhokelo soPhuculo lovavanyo lokuhambahamba kwecala
Uvavanyo lweLateral Flow Assay sisixhobo esisetyenziselwa uvavanyo olukhawulezileyo ekhaya kunye novavanyo lweklinikhi. Zezona zixhobo zithembekileyo nezichanekileyo ezisetyenziswa phantse kuwo onke amazwe aphuhlileyo nasaphuhlayo. Kwakhona, iSikhokelo soPhuhliso lweLateral Flow Assay sinamanyathelo aphantse abe li-10 amisela inkqubo yophuhliso lweLateral Flow Assay. Ngokwesiqhelo, iiLateral Flow Assays zinesampulu yephedi, i-conjugate pad, i-nitrocellulose membrane, iilebhile ze-adsorbent pad, kunye ne-buffer. Nangona kunjalo, kunokubakho utshintsho oluthile kwiilebhile njengoko kukho iilebhile ezahlukeneyo ezisetyenziswa kwii-lateral flow assays.
Ukuze uqalise isikhokelo sophuhliso lwe-lateral flow assay , kubalulekile ukuqwalasela amanyathelo ambalwa ngaphambi kokuba uqale inkqubo. Okokuqala, kubalulekile ukuhlalutya ifomathi eza kukhethwa kwi-lateral flow assay nokuba ingaba yifomathi yokhuphiswano okanye yesandwich. Ngokusisiseko, isikhokelo sophuhliso lwe-Lateral flow Assay sixhomekeke kwinkqubo yophuhliso lwe-assay. Inyathelo lokuqala elibalulekileyo nelibaluleke kakhulu kukuchonga i-control assay eya kunceda ukuchonga i-target analyte kwisampulu. Esi sikhokelo sophuhliso siya kunika inkqubo yophuhliso lwe-LFA ngokubanzi kuba i-assays ezahlukeneyo zinenkqubo eyahlukileyo.
Inyathelo lokuqala kwisikhokelo sophuhliso kukukhethwa kwee-nanoparticles. Ukukhethwa kwee-nanoparticles kuyahluka ngobukhulu kunye nokuma. Kukho ubungakanani obahlukeneyo obufumanekayo kwiimvavanyo zokuhamba kwecala. Kukwakho nee-nanoparticles zegolide ezingama-40nm ezine-carbon okanye umphezulu we-citrate onokusetyenziswa ngakumbi kwinkqubo yokufunxa iiproteni. Ezinye ii-nanoparticles ezinokukhethwa kuphuhliso lwe-LFA ziquka i-carboxyl gold, ii-nanoshells zegolide ze-carboxyl, njl.njl. ngaphandle koko kukho ezinye ii-probes ezifana nee-cellulose beads, i-polystyrene edayiweyo, kunye nee-fluorescent probes. Ingongoma entle yokukhetha ii-nanoparticles zegolide ze-carboxyl kukuba kwinkqubo yokufunxa kufuneka ii-antibodies ezimbalwa kakhulu kwaye ngenxa yezi nanoparticles kwakheka i-bond eyi-amide bond ezinzileyo nengaguqukiyo. Nangona kunjalo, kufuneka kukhunjulwe ukuba amandla avelayo afumaneka phakathi kwee-nanoparticles kunye nee-antibodies abangelwa yindawo yazo yokudibanisa.
Inyathelo lesibini kukuKhethwa kweAntibody. Ukhetho lwe-Antibody linyathelo elibalulekileyo njengoko ukusebenza kwe-LFAs kuxhomekeke kubumbano lwee-antibodies, kinetics, kunye nezinye iipropathi. Kwakhona, ukubopha ii-antibodies kubalulekile ekufumaneni iziphumo. Enye into ebalulekileyo ngelixa ukhetha ii-antibodies ziindidi zazo ezimbini. Ezi zimbini iintlobo ze-antibodies zi-monoclonal kunye ne-polyclonal. Iintsholongwane zeMonoclonal kulula ukuzenza ngelixa ii-polyclonal antibodies zinobuntununtunu obuphezulu ngokuthelekiswa ne-monoclonal. Kwinqanaba lenkqubo yokhetho lomzimba, kukho amanye amanyathelo amabini okt ukucocwa kwe-antibody, kunye nolawulo lwe-antibody yomgca. Kukhetho lwe-antibody, isiqu esicetyiswayo kukuhlola ii-antibodies ngesizathu sokuba kufuneka kuvavanywe kwangaphambi kwexesha. Amachiza omzimba anokuvavanywa kwaye avavanywe ngokwakha uhlobo lokuqala kunye nolokuqala lokuvavanywa kokuhamba komva okunokusebenza njengesixhobo sesampulu yovavanyo. Ukusebenza kwee-antibodies kwahlukile kuhlobo olwahlukileyo lwee-LFA. Umzekelo, kwi-ELISA i-kinetics ayibalulekanga kangako. Kwaye xa uthetha nge-assal flow flow, kukho iimeko ezithile ekufuneka ii-antibodies zikwazi ukuzenza. Okokuqala, ii-antibodies kufuneka zisebenze nasemva kokuba zidibene neenanoparticles kwaye ukuthembeka kwazo kulwakhiwo akufuneki kwenzakaliswe nangayiphi na indlela. Kananjalo, kufuneka babenako ukunika impendulo yabo ngoko nangoko xa isampulu iphinda iphinde ibuyiselwe kwisampulu. Ixesha lokuxubusha kwezinye iintlobo zovavanyo lide xa kuthelekiswa noVavanyo oluValiweyo oluhamba mva apho ukubophelela kulayini wovavanyo kufuneka kwenziwe ngaphakathi kwexesha lemizuzwana embalwa.
Inkqubo yesithathu kule nkqubo yimbumba ye-nitrocellulose kwaye iyinxalenye ebaluleke kakhulu ye-LFA njengoko inemigca yovavanyo kunye nolawulo. Inwebu ye-nitrocellulose inezinto zokusebenza ezisebenza ukuvelisa iziphumo. Iyafumaneka ngobukhulu obahlukeneyo, i-porosities, kunye noyilo. Ngokwenyani, inwebu ye-nitrocellulose ixhomekeke kwixesha lokuhamba kwe-capillary. Ngexesha lokuhamba kwe-capillary, oko kuthetha ukuba ixesha elifunekayo kwisampulu ukuba liye kwi-4cm. Kweli nyathelo, kukho elinye inyathelo elibizwa ngokuba kukuhluba inwebu. I-buffer apho iprotheyini ethile enemigca inokuba nefuthe elibonakalayo kwiziphumo zokugqibela zalo naluphi na uvavanyo lokuhamba okulandelayo. Nangona iiprotheyini ezininzi zinokuchithwa kwi-1x PBS, ezinye zinobuzaza kakhulu kwi-pH, uxinzelelo lwetyuwa, kunye nokuvela kwesixazululi esinokuthiywa ngegama i-trehalose. Ukuhluba inwebu lelinye inyathelo elibalulekileyo eliza neli nyathelo. Kweli nqanaba, uyilo lovavanyo lokuvavanywa kokuhamba ngokulandelelana kukroba uvavanyo kunye nokulawula imigca ye-antibody ekhoyo kwi-membrane ye-nitrocellulose. Ukuqwalaselwa okubalulekileyo kweli nyathelo kukwimbumba ye-nitrocellulose apho kusithiwa ayigcini inwebu ye-nitrocellulose kwindawo apho kungekho kufuma kunokuba yomile. Ukuphuhliswa kwe-assay yokuhamba kwecala, iqondo lobushushu lenwebu ye-nitrocellulose kufuneka ifumile. Njengoko kukho iifomathi ezahlukeneyo zokuvavanywa, okt ifomathi yokhuphiswano kunye nefomathi yesandwich nayo ebaluleke kakhulu. Uxinzelelo lwe-antibody luyahluka kuzo zombini iifomati zefomathi yesandwich uxinzelelo lwe-antibody olucetyiswayo yi-1mg / mL yovavanyo kunye nolawulo lwe-antibodies yomgca kuphela kwaye lo mda unokufikelela kwi-0.5 ukuya kwi-2 mg / mL. Kwinyathelo le-nitrocellulose membrane kunye nesikhokelo sokukhula kokuhamba, elinye inyathelo elibandakanyiweyo linyathelo lokuthintela inwebu. Ukwenza eli nyathelo kunezibonelelo ezininzi ezinjengokuphucuka kokuhamba kolwelo, ukuzinza kwemigca yovavanyo, kunye nokuthintela ukungangqinelani okubophelelayo kunika iziphumo eziyinyani. Uninzi lwenwebu ye-nitrocellulose kwi-assal flow flow assay, kukho isisombululo esichaphazelekayo esongezwa kuyo ngokwenza inwebu ye-hydrophilic.
Inyathelo elilandelayo kukudibanisa kunye nokudibanisa iphedi. Ukuphuhlisa uvavanyo lwecala lokuhamba ngokulandelelana kubalulekile ekuphuhliseni ukwenziwa kokudibanisa iiparameter. Xa i-conjugate ilungiselelwe i-assortal ye-lateral flow ikhutshwa kwaye yomiswe kwi-pad conjugate. Ukuphuhlisa ukudibanisa okuzinzileyo kwee-antibodies kunokubakho amanyathelo aliqela. Amanyathelo abandakanyekayo ekuphuhliseni ukusebenza ngokudibeneyo kunye nokuphuculwa kokudibana. Kweli nqanaba, uzinzo lokudibana linyathelo elibaluleke kakhulu kwaye ke, uzinzo lwecolloidal kufuneka luphuculwe ngelixa kuyilwa i-LFA. uzinzo lwe-colloidal lubaluleke kakhulu kulingo lokuhamba ngokulandelelana kwaye ke ngelixa kuyilwa kunye nokwenza ngcono i-LFA oku kuhlala kugcinwa kuthathelwa ingqalelo. Ii-nanoparticles zegolide ezidlala indima ebalulekileyo ekuhlanganiseni kwi-LFA zineempawu ezibonakalayo ezikhethekileyo ngoncedo lokufunxa nokusasaza ukukhanya ngokuchanekileyo nangokufanelekileyo. Unxibelelwano olomeleleyo phakathi kwee-nanoparticles kunye nokukhanya kwenzeka ngenxa yee-elektroniki zokuhambisa ezikhoyo kumphezulu wesinyithi okhokelela ekusongeleni ngokudibeneyo xa ii-elektroni zivuya kukukhanya ngokuhamba rhoqo kunye nokulingana. Emva kokuba kukho indibaniselwano eyimpumelelo kukho iziphumo ezithile ezibonakalayo kwiimpawu ezibonakalayo njengoko utshintsho lubonwa kwisalathiso sokuphinda sibonwe ngakumbi kuluhlu lwe-UV-vis ngokwahlula umgca obomvu. Kananjalo kunyango lwephedi yokudibanisa, kunokuba neendlela ezahlukeneyo. Ezinye zazo kukuntywiliselwa, okanye ukutshiza ngokufanayo ngoncedo lwesixhobo esizisebenzelayo.
Emva koko, inkqubo yokukhetha iphedi yesampulu iyenzeka. Le yinxalenye yokuqala yovavanyo lokuhamba kwesampulu , ngoko ke kubalulekile ukujonga izinto zephedi yesampulu kunye nonyango lwangaphambi konyango lwephedi yesampulu ukuze kubonwe ukuhamba kwayo kunye nokusebenza kwayo. Iiphedi zesampulu ezisetyenziselwa uvavanyo lokuhamba kwesampulu zeentlobo ezahlukeneyo ezifana nefayibha, umqhaphu, i-synthetic, njl. Kuye kwabonwa ukuba ezinye zeesampulu ezifana nomchamo, igazi, njl. zinokutshintsha ngenxa yezinye izinto ezininzi ezibandakanya izinto zebhayoloji, ixesha lokutya, isiselo esithathwayo ngaphambi kokuba kuqokelelwe isampuli kwisixhobo sovavanyo lokuhamba kwesampulu, kunye nezinye ezininzi. Ukunyanga iphedi yesampulu kunye nokuyiphucula, i-buffer elungiselelweyo ingasetyenziswa, ekwanazo neenzuzo ezininzi. Ngelixa kuphuhliswa uvavanyo lokuhamba kwesampulu, kunye nokukhetha iphedi yesampulu, i-buffer eyenziwe nge-ootmised inokunceda ukuphucula ukusebenza kovavanyo ngokunciphisa ukuguquguquka kwesampulu equka iproteni, i-pH, i-viscosity, uxinzelelo lwetyuwa, njl. Ingaphucula nokuhamba kunye nokuhambelana kovavanyo ngelixa luphuhliswa. Ii-buffers zonyango zibonakaliswe ziluncedo kakhulu ekugcineni ukuhamba kunye nokuhambelana, zilungisa i-pH, zisebenza njengee-blockator agents ngokudibanisa iiproteni, iityuwa, ii-surfactants kunye noxinzelelo olufanelekileyo lwento nganye. Eyona nkcazelo ibalulekileyo yesampulu yokujonga kumthamo wokufunxa kuba oko kuthetha malunga nomthamo wesampulu yovavanyo.
Emva kokuba iphedi yesampulu ikhethiwe ukhetho lwepick pad luza kulandela. Iphedi yokujija inceda kwinkqubo ye-adsorption yezixhobo ezingathathwanga nangayiphi na indlela okt umgca wovavanyo kunye nolayini wokulawula. Iphedi yokujija ikwanqanda umva wokubuyela umva kolwelo olusetyenziswa njengesampulu. Kucetyiswa ukuba ngelixa ukhetha umbandela wokujija izinto kufuneka zihlolwe ukugcina isixhobo nakuphi na ukungangqinelani okunokukhokelela kwimicimbi kwaye mhlawumbi neziphumo ezingezizo. Ngaphandle kwesiqhelo, izinto ezityebileyo okanye iphedi yokujija ayingcono kunaleyo icekeceke. Izinto ezinobuncinci zibhetele ekwenzeni ngcono kwaye zihlala zinezinto eziphezulu ngamanye amaxesha. Kodwa yonke into kufuneka ihlolwe kwiimpawu zayo kunye nokufaneleka kwayo kuphuhliso lwe-assay yecala lokuhamba. Ukukhetha ubungakanani kunye nezinto zephedi yokujija kuyafuneka ukuyigcina ilungelelaniswe nento yokuba amandla okufunxa kufuneka abe ngaphezulu kwaye umthamo we-buffer kufuneka usebenze.
Kwi-assal ye-lateral flow, indibano yovavanyo lwe-strip ikwalinyathelo lesikhokelo sayo sophuhliso. Kwindibano yovavanyo lwe-strip, ii-LFA zihlanganisiwe ngokusekwe kwimveliso enkulu okanye kwimveliso encinci. Kwezinye iintlobo zokuvavanywa kokuhamba ngokulandelelana, ubungakanani bokufundwa ngaphandle kunokuba kungafuneki kodwa ifomathi yediploma iya kufuneka ekusebenziseni indibano yovavanyo lwe-strip kwi-LFA. Kukho ikhasethi enoluvavanyo lomtya ngaphakathi lelinye lawona manyathelo abaluleke kakhulu kwaye abaluleke kakhulu ekufezekiseni i-assay enokuphinda iveliswe kwakhona kwaye ithembeke. Ukucaciswa okubini okukhankanywe ngaphambili kubaluleke kakhulu kuvavanyo lobungakanani, ngakumbi. Owona msebenzi uphambili wekhasethi kukubonelela ngoxinzelelo olufunekayo kulwelo. Oku kukuqinisekisa ukuba kukho ukuhamba ngokugqibeleleyo kwaye ulwelo luyadlula kwindibano kwinto efanayo. Ikhasethi ikwaqinisekisa ukuba ulwelo aluhlali neziphumo emaphethelweni kwaye luhamba nje kwiipads.
Emva kwendibano yovavanyo lwe-strip, inyathelo elilandelayo liqhuba uvavanyo. Kukho iindlela ezahlukeneyo zokuqhuba i-assay ezinje nge-assay yesithili kwi-conjugate engamanzi, uvavanyo olupheleleyo lwe-conjugate eyomileyo, kunye ne-buffer esebenzayo. Uhlalutyo lomtya, kufuneka yaziwe ukuba ngaba uvavanyo lusezingeni okanye luchanekile kwaye kufuneka kukhethwe indlela eyiyo yohlalutyo. Ukuhlalutya i-assay, inokwenziwa ngeliso lenyama apho kuya kubakho ewe / akukho ziphumo okanye ngokufaka umbono womgca wovavanyo, iskena seflatbed okanye ikhamera enokukhanya okulawulwayo inokusetyenziswa. Inyathelo lokugqibela kukuphuculwa kwesilingo ekukhetheni isibini esichasene ne-antibody, initrocellulose membrane, umsonto we-wick, iphedi ye-adsorbent, nawo onke amanye amalungu afakiwe.
Isiphelo:
Isikhokelo sophuhliso lweLateral Flow Assay sinamanyathelo alithoba ukuya kwalishumi anikiweyo ekufuneka alandelwe ngocoselelo ukwenza uvavanyo lwe-lateral flow.
Ucaphulo
https://cdn.shopify.com/s/files/1/0257/8237/files/BioReady_Lateral_Flow_Handbook.pdf