Chitsogozo Chotsatira Chakuyenda kwa Assay
Kuyesa kwa Mayendedwe a M'mbali ndi chipangizo chomwe chimagwiritsidwa ntchito poyesa mwachangu kunyumba komanso poyesa zachipatala. Ndi zipangizo zodalirika komanso zolondola kwambiri zomwe zimagwiritsidwa ntchito pafupifupi m'maiko onse otukuka ndi otukuka. Komanso, Buku Lophunzitsira Kukula kwa Mayendedwe a M'mbali lili ndi masitepe pafupifupi 10 omwe amatsimikiza njira yopangira Mayendedwe a M'mbali. Nthawi zambiri, Mayendedwe a M'mbali amakhala ndi sample pad, conjugate pad, nitrocellulose membrane, ma label adsorbent pad, ndi buffer. Komabe, pakhoza kukhala kusintha kwina m'ma label chifukwa pali ma label osiyanasiyana omwe amagwiritsidwa ntchito mu ma lateral flow assays.
Kuti muyambe buku lotsogolera pakupanga mayeso a lateral flow assay , ndikofunikira kuganizira zinthu zochepa musanayambe ntchitoyi. Choyamba, ndikofunikira kusanthula mawonekedwe omwe asankhidwa pa mayeso a lateral flow kaya ndi mpikisano kapena mtundu wa sandwich. Kwenikweni, buku lotsogolera pakupanga mayeso a lateral flow Assay limadalira njira yopangira mayeso. Gawo loyamba lofunikira komanso lofunika kwambiri ndikupeza mayeso owongolera omwe angathandize kuzindikira analyte yomwe ili mu chitsanzo. Buku lotsogolerali lidzapereka njira yonse yopangira LFA chifukwa mayeso osiyanasiyana ali ndi njira yosiyana.
Gawo loyamba la chitsogozo cha chitukuko ndi kusankha tinthu tating'onoting'ono. Kusankha tinthu tating'onoting'ono timasiyana kukula ndi mawonekedwe. Pali kukula kosiyanasiyana komwe kulipo poyesa kuyenda kwa lateral. Palinso tinthu tating'onoting'ono ta golide ta 40nm tokhala ndi kaboni kapena citrate pamwamba zomwe zingagwiritsidwenso ntchito popititsa patsogolo njira yopezera mapuloteni. Tinthu tating'onoting'ono ta golide ta 40nm tomwe tingasankhidwe popanga LFA ndi tinthu tating'onoting'ono ta golide ta carboxyl, tinthu tating'onoting'ono ta golide ta carboxyl, ndi zina zotero. Kupatula apo pali ma probe ena monga mikanda ya cellulose, polystyrene yopaka utoto, ndi ma probe a fluorescent. Ubwino wa kusankha tinthu tating'onoting'ono ta golide ta carboxyl ndikuti pa njira yopezera ma antibodies ochepa amafunika ndipo chifukwa cha tinthu tating'onoting'ono ta golide ta carboxyl timapangidwa kukhala mgwirizano wokhazikika komanso wosasinthika wa amide. Komabe, tiyenera kukumbukira kuti mphamvu zomwe zimapezeka pakati pa tinthu tating'onoting'ono ta golide ndi ma antibodies zimachokera ku malo awo olumikizirana.
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 yosankha chitsanzo cha pad imachitika. Iyi ndi gawo loyamba la kuyesa kwa lateral flow kotero ndikofunikira kuyang'ana zinthu za pad ya chitsanzo ndi kukonza pad ya chitsanzo kuti muwone ngati ikuyenda bwino komanso momwe imagwirira ntchito. Zitsanzo za pad zomwe zimagwiritsidwa ntchito poyesa lateral flow ndi zamitundu yosiyanasiyana monga ulusi, thonje, zopangidwa, ndi zina zotero. Zawonedwanso kuti zitsanzo zina monga mkodzo, magazi, ndi zina zotero zimatha kusiyanasiyana chifukwa cha zinthu zina zambiri zomwe zimaphatikizapo zinthu zamoyo, nthawi yodyera, chakumwa chomwe chatengedwa musanatenge chitsanzocho pa chipangizo choyesera lateral flow, ndi zina zambiri. Pochiza chitsanzo cha pad ndikuchikonza bwino, buffer yokonzedwa ingagwiritsidwe ntchito yomwe ili ndi zabwino zambiri. Popanga mayeso a lateral flow, ndikusankha chitsanzo cha pad, buffer yopangidwa ndi ootmised ingathandize kupititsa patsogolo magwiridwe antchito a mayeso pochepetsa kusinthasintha kwa chitsanzo chomwe chimaphatikizapo mapuloteni, pH, viscosity, kuchuluka kwa mchere, ndi zina zotero. Ikhozanso kusintha kuyenda ndi kusinthasintha kwa mayeso pamene ikupangidwa. Ma buffers ochizira awonetsedwa kuti ndi othandiza kwambiri pakusunga kuyenda ndi kusinthasintha, kupangitsa pH kukhala yabwinobwino, kugwira ntchito ngati zoletsa mwa kuphatikiza mapuloteni, mchere, ndi ma surfactants ndi kuchuluka koyenera kwa chilichonse. Chofunika kwambiri pa chitsanzo cha pad kuti muwone ndi mphamvu ya kuyamwa chifukwa chimafotokoza za kuchuluka kwa chitsanzo cha mayeso.
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:
Buku lotsogolera pakupanga mayeso a Lateral Flow Assay lili ndi njira zisanu ndi zinayi mpaka khumi zomwe ziyenera kutsatiridwa bwino popanga mayeso a lateral flow.
Zothandizira
https://cdn.shopify.com/s/files/1/0257/8237/files/BioReady_Lateral_Flow_Handbook.pdf