Chitsogozo Chotsatira Chakuyenda kwa Assay
Otsatira Otsatira Assay ndi chida chomwe chimagwiritsidwa ntchito poyesa mwachangu kunyumba komanso kuyezetsa kuchipatala. Ndiwo zida zodalirika komanso zolondola zomwe zikugwiritsidwa ntchito pafupifupi mayiko onse otukuka komanso akutukuka. Komanso, ofananira otaya Yesani Chitsogozo Chachitukuko ili ndi masitepe pafupifupi 10 omwe amatsimikizira njira yopangira Lateral Flow Assay. Kawirikawiri, Kufufuza Kwotsatira khalani ndi phukusi loyeserera, conjugate pad, nitrocellulose membrane, zolemba adsorbent pad, ndi buffer. Komabe, pakhoza kukhala kusintha pamakalata popeza pali zolemba zosiyanasiyana zomwe zimagwiritsidwa ntchito poyesa kutuluka.
Kuyamba malangizo owongolera otsogola, ndikofunikira kuganizira zochita zingapo musanayambe ntchitoyi. Choyamba, ndikofunikira kuwunika mtundu womwe ungasankhidwe pakuyesa kotsatira ngati kungakhale mpikisano kapena sangweji. Kwenikweni, chiwongolero chakuwongolera kwa Lateral flow Assay chimadalira njira yakukula kwamayeso. Gawo loyamba lofunikira komanso lofunikira kwambiri ndikuzindikira zoyeserera zomwe zingakuthandizeni kuzindikira omwe akuwunikira mu zitsanzozo. Kuwongolera uku kukupatsani njira yachitukuko ya LFA chifukwa mayesedwe osiyanasiyana amakhalanso ndi njira ina.
Gawo loyamba ku kalozera chitukuko ndiye kusankha kwa nanoparticles. Kusankhidwa kwa nanoparticles kumasiyanasiyana kukula ndi mawonekedwe. Pali mitundu yosiyana siyana pamayeso oyenda pambuyo pake. Palinso ma nanoparticles agolide a 40nm okhala ndi kaboni kapena citrate pamwamba omwe amatha kugwiritsidwanso ntchito popanga mapuloteni. Zina mwa ma nanoparticles omwe angasankhidwe kuti apange chitukuko cha LFA ndi carboxyl golide, carboxyl nanoshells agolide, ndi zina zambiri kupatula kuti pali ma probes ena monga ma cellulose mikanda, utoto wa polystyrene, ndi ma fluorescent probes. Chowonjezera pamasankhidwe a carboxyl golide nanoparticles ndikuti pakufalitsa ma adsorption pamafunika ma antibodies ochepa kwambiri chifukwa cha ma nanoparticles chomangira chimakhala cholimba komanso chosasinthika chomangira amide. Komabe, ziyenera kukumbukiridwa kuti mphamvu zomwe zimapezeka pakati pa nanoparticles ndi ma antibodies zimachitika chifukwa cholumikizana.
Gawo lachiwiri ndikusankhidwa kwa Antibody. Kusankha ma antibody ndi gawo lofunikira chifukwa magwiridwe antchito a LFAs zimatengera kuyandikira kwa ma antibodies, kinetics, ndi zina zotengera. Komanso, kumangiriza ma antibodies ndikofunikira pakuzindikira zotsatira. China chofunikira posankha ma antibodies ndi mitundu iwiri ya iwo. Mitundu iwiri ya ma antibodies ndi monoclonal komanso polyclonal. Ma antibodies a monoclonal ndiosavuta kupanga pomwe ma antibodies a polyclonal amakhala ndi chidwi chachikulu poyerekeza ndi monoclonal. Mu gawo la kusankha kwa antibody, palinso njira zina ziwiri monga kuyeretsa kwa antibody, ndi mzere wazowongolera. Posankha ma antibodies, tsinde lomwe lanenedwa ndikuwunika ma antibodies chifukwa choti amayesedwa kale. Ma antibodies amatha kuwunikidwa ndikuyesedwa pomanga mtundu woyambirira komanso woyambirira wa kuyesa kwakanthawi komwe kumatha kugwira ntchito yoyeserera. Magwiridwe anthawi zonse amtundu wa ma LFAs. Mwachitsanzo, ku ELISA kinetics siyofunika kwenikweni. Ndipo polankhula za mayendedwe ofananira nawo, pali zinthu zina zomwe ma antibodies amayenera kuchita. Choyambirira, ma antibodies amayenera kugwira ntchito ngakhale atalumikizidwa ndi nanoparticles ndipo mawonekedwe awo sayenera kuvulazidwa mwanjira iliyonse. Komanso, ayenera kukhala ndi kuthekera kofulumira kuyankha momwe chitsanzocho chilimbikitsidwanso ndi chitsanzocho. Nthawi yophatikizira mu mitundu ina ya Assays ndiyotalikirapo poyerekeza ndi Lateral Flow Assay momwe kumangiriza pamzere woyeserera kuyenera kuchitidwa mkati mwa nthawi yayitali yamasekondi ochepa.
Njira yachitatu pakuchita izi ndi nembanemba ya nitrocellulose ndipo ndi gawo lofunikira kwambiri ku LFA popeza ili ndi mizere yoyesa ndikuwongolera. Kakhungu ka nitrocellulose kamakhalanso ndi ma reagents omwe amagwira ntchito kuti apange zotsatira. Imapezeka mosiyanasiyana, mosiyanasiyana, ndi kapangidwe kake. Kwenikweni, nembanemba ya nitrocellulose imadalira nthawi yoyenda ya capillary. Pogwiritsa ntchito nthawi ya capillary, zikutanthauza kuti nthawi yomwe mayesowo amayenera kutsikira mpaka 4cm. Pachigawo ichi, pali gawo lina lomwe limatchedwa kuchotsa nembanemba. Chikhazikitso chomwe mapuloteni ena amakhala ndi mizere chimatha kukhala ndi gawo lalikulu pamapeto omaliza oyeserera ofananira nawo. Ngakhale mapuloteni ambiri amatha kusungunuka mu 1x PBS, ena amakhudzidwa kwambiri ndi pH, mchere wamchere, komanso kupezeka kolimba komwe trehalose lingatchulidwe. Kuchotsa Kakhungu ndi gawo lina lofunikira lomwe limabwera ndi gawo ili. Pakadali pano, kapangidwe kake koyeserera koyeserera ndikumayesa mayeso ndikuwongolera mizere ya antibody yomwe ilipo pa nembanemba ya nitrocellulose. Chofunika kwambiri pagawo ili ndikutulutsa kwa nitrocellulose nembanemba komwe akuti sikungasunge nembanemba ya nitrocellulose pamalo pomwe kulibe chinyezi m'malo mwake yonse imawuma. Pakukula kwa mayendedwe ofananira nawo, kutentha kwa nembanemba ya nitrocellulose kuyenera kukhala chinyezi. Popeza pali mitundu yosiyanasiyana ya zofufuza, mwachitsanzo mtundu wopikisana ndi mtundu wa sangweji zomwe ndizofunikanso kwambiri. Magulu a antibody amasiyana pamitundu yonse ya sangweji yomwe mankhwala oyeserera a 1mg / mL amangoyesa ndi kuwongolera ma antibodies am'malire ndipo malirewa amatha mpaka 0.5 mpaka 2 mg / mL. Mu gawo la nitrocellulose nembanemba ndikuwongolera kotsatira kwa njira yolowera, gawo lina lomwe likuphatikizidwa ndi gawo lotsekereza nembanemba. Kuchita izi kuli ndi maubwino ambiri monga kuyenda bwino kwa madzimadzi, kukhazikika kwamayeso, ndikuletsa kumangako kosakhala kwenikweni kumapereka zotsatira zowona. Pambiri ya nembanemba ya nitrocellulose pakayesedwe kotsatira, pali njira yothandizira yomwe imawonjezeredwa pakupanga nembanemba hydrophilic.
Gawo lotsatira ndikuphatikizira ndi conjugate pad. Pakukhazikitsa njira yoyeserera yofunikira ndikofunikira kukhazikitsa magawo ophatikizira. Pomwe conjugate imakonzedwa kuti ikayendetsedwe kotsatira imagawidwa ndikuwuma pa phukusi la conjugate. Pakakhala njira zingapo zothanirana ndi ma antibodies. Masitepe omwe akukhudzidwa pakupanga mgwirizano wolimbikitsana. Pakadali pano, kukhazikika kwa conjugation ndichinthu chofunikira kwambiri, chifukwa chake, kukhazikika kwa colloidal kuyenera kukonzedwa bwino pakupanga LFA. kukhazikika kwa colloidal ndikofunikira kwambiri pakuyesa kotsalira koteroko motero popanga ndikukhathamiritsa LFA izi zimasungidwa nthawi zonse. Ma nanoparticles agolide omwe amatenga gawo lofunikira pakulumikizana ku LFA ali ndi mawonekedwe apadera kwambiri mothandizidwa ndi omwe amalowetsa ndikuwunika kuyatsa molondola komanso moyenera. Kulumikizana kwamphamvu pakati pa ma nanoparticles ndi kuwala kumachitika chifukwa cha ma elekitironi omwe amapezeka pamwamba pazitsulo zomwe zimapangitsa kuti kusokonekera palimodzi pamene ma elekitironi amasangalatsidwa ndi kuwala mofanana komanso pafupipafupi. Pakakhala kulumikizana kopambana pamakhala zovuta zina pazowoneka bwino chifukwa kusintha kumawonedwa mu index ya refractive yomwe imatha kutsatiridwa ndi mawonekedwe a UV-vis ndi kusiyana kwa mzere wofiira. Komanso pochizira phukusi lolumikizirana, pakhoza kukhala njira zosiyanasiyana. Ena mwa iwo ndimiza, kapena kupopera yunifolomu mothandizidwa ndi makina okhala ndi makina.
Pambuyo pake, njira zosankhira pad zimachitika. Ili ndiye gawo loyamba la otaya ofananira nawo kuyesa ndiye kuti ndikofunikira kuwunika zitsanzo za pad ndi chithandizo chisanachitike chazitsulocho ngati chikuyenda bwino. Mitundu yoyeserera yomwe imagwiritsidwa ntchito poyeserera mosiyanasiyana ndi yamitundu yosiyanasiyana monga fiber, thonje, zopangira, ndi zina zambiri. Phatikizani zinthu zamoyo, kudya nthawi, zakumwa zomwe zidatengedwa musanatenge nyemba pazida zoyeserera, ndi zina zambiri. Pochizira pulogalamuyo ndikuyikhathamiritsa chikhomo chomwe chingagwiritsidwe ntchito chomwe chingakhale ndi maubwino ambiri. Pomwe mukuyesa kuyesa kwa lateral, ndikusankha sampuli ya ootmized buffer kumatha kuthandiza kupititsa patsogolo kuyesaku pochepetsa kusintha kwa nyemba zomwe zimaphatikizapo mapuloteni, pH, mamasukidwe akayendedwe, kuchuluka kwa mchere, ndi zina zambiri. kuyenda ndi kusasinthasintha kwa mayesedwe pamene akupangidwa. Chithandizo cha buffers chatsimikiziridwa kuti ndi chothandiza kwambiri pakusunga mayendedwe ndi kusasinthasintha, kuimitsa pH, kugwira ntchito ngati zotchingira polumikizana ndi mapuloteni, mchere, opanga ma surfactant omwe ali ndi magulu oyenera a aliyense. Chofunikira kwambiri pamtengo woyeserera ndi kuchuluka kwa mayamwidwe chifukwa zimafotokoza za voliyumu yoyeserera.
Pambuyo pachithunzicho atasankhidwa kusankha kwa chingwe cha wick kumabwera motsatira. Padi yolumikizira imathandizira pakutsatsa kwa ma reagents omwe sanatengeke ndi mzere uliwonse mwachitsanzo mzere woyeserera ndi mizere yolamulira. Phukusi loyimitsalo limayimitsanso madzi obwerera kumbuyo omwe amagwiritsidwa ntchito ngati zitsanzo. Akuti posankha zinthu zolembera pakhomopo, zinthuzo ziyenera kufufuzidwa kuti zisungire chipangizocho pazosiyana zilizonse zomwe zingayambitse mavuto ndipo mwina zotsatira zabodza. Zina kuposa zachilendo, zakuda kapena chopukutira sichabwino kuposa chopyapyala. Zowonda ndizabwino kuchita bwino ndipo nthawi zambiri zimakhala ndi zida zapamwamba nthawi zina. Koma chilichonse chiyenera kufufuzidwa ngati chili ndi mawonekedwe ake komanso kuyenerana kwawo pakupanga njira yoyeserera yoyenda. Posankha kukula ndi zinthu zolembedwazo pamafunika kuti zizigwirizana ndi zomwe zimafunika kuti mayamwidwe ake akhale okwera ndipo voliyumu yoyenda iyenera kuti ikuyenda.
Pazoyeserera zoyandikira, msonkhano woyesa mzere ndi gawo limodzi la chiwongolero cha chitukuko. Pamsonkhano woyeserera, ma LFA asonkhanitsidwa kutengera kupanga kwakukulu kapena kupanga pang'ono. Kwa mitundu ina yamayeso oyenda mozungulira, kuwerengera kochulukirapo sikungakhale kofunikira koma mawonekedwe a dipstick angafunike poyendetsa msonkhano woyeserera ku LFA. Pali kaseti yomwe ili ndi mayeso amkati mkati ndi imodzi mwanjira zofunika kwambiri komanso zofunika kwambiri kuti mukwaniritse zofunikirazo zomwe ndizoberekanso komanso zodalirika. Malingaliro awiri omwe atchulidwa kale ndi ofunikira kwambiri pakuyesa kwamankhwala, makamaka. Ntchito yayikulu ya kaseti ndikupereka kukakamizidwa kumadzi. Izi ndikuwonetsetsa kuti pali kutuluka koyenera ndipo madziwo akudutsanso pamsonkhano womwewo. Kasetiyo imawonetsetsanso kuti madziwo satsatira zotsatira zake m'mphepete mwake ndikungoyenda pamapepala.
Pambuyo pamsonkhano woyeserera, gawo lotsatira likuyesa. Pali njira zosiyanasiyana zoyeserera poyeserera monga kuyesa kwa chigawo mu conjugate yamadzimadzi, kuyesa kwathunthu kwa conjugate yowuma, ndi gawo lotetezera. Pofufuza mzerewo, ziyenera kudziwika kuti ngati mayesowo ndi oyenera kapena owerengeka ndipo njira yoyenera kuwunika iyenera kusankhidwa. Kuti tiwunikire mayeso, zitha kuchitidwa kudzera m'maso momwe mungakhale inde / ayi zotsatira kapena kujambula chithunzi cha mzere woyeserera, chojambulira cha flatbed kapena kamera yokhala ndi kuyatsa kogwiritsidwa ntchito itha kugwiritsidwa ntchito. Gawo lomaliza ndikuthandizira kuyesa komwe amasankha ma antibody awiri oyenera, nembanemba ya nitrocellulose, chingwe chachingwe, phukusi la adsorbent, ndi magawo ena onse akuphatikizidwa.
Kutsiliza:
The chitsogozo chachitukuko cha Lateral Flow Assay Pali njira zisanu ndi zinayi mpaka khumi zomwe ziyenera kutsatiridwa bwino popanga kuyeserera kotsatira.
Zothandizira
https://cdn.shopify.com/s/files/1/0257/8237/files/BioReady_Lateral_Flow_Handbook.pdf