Nzvimbo Yokudzivirira Mhuka

Manyuko emhuka eAlpha Lifetech akajeka, ane mvumo yekuongorora nekuisa vanhu muchivande, mamiriro ekuberekwa akajeka, nguva dzakajeka dzekubaiwa majekiseni, uye zvinyorwa zvakadzama zvenguva dzekubaiwa majekiseni, zvichipa masevhisi ari nyore ekugadzira maantibody. Inonyanya kubaiwa majekiseni mhuka dzeCamelidae (Alpaca Liama, neCameras), ichiisa hwaro hwekugadzirwa kwe nanobody. Pamusoro pezvo, inogona zvakare kubaiwa majekiseni mhuka dzakajairika (dzakadai setsuro chena dzeNew Zealand, mbeva, nedzimwe mhuka) zvichienderana nezvinodiwa nevatengi zvekubaiwa majekiseni uye zvinodiwa zvekudzivirira muviri, kuabvisa nekuaparadzanisa muPBMCs, uye kuashandisa pakuedza kunotevera (senge monoclonal antibody, polyclonal antibody development, nezvimwewo). Vatengi vanogona kupa ma immunogens avo, uye tichaita kudzora kwakasimba kwemhando paari. Kana vatengi vakapa ma immunogens ehutachiona, anofanira kuvharwa uye tichaita bvunzo dzekubaiwa majekiseni kuti tive nechokwadi chekuti akachengeteka. Panguva imwe chete, tinogonawo kugadzirisa ma immunogens anoenderana zvichienderana nezvinodiwa nevatengi.

Nhanganyaya yekudzivirira mhuka

Pakugadzira ma antibodies anobva kumhuka akadai se monoclonal antibodies, polyclonal antibodies, uye nanobodies, danho rekutanga nderekubaya mhuka uye kuwana masero eB, ma antibodies, kana maPBMC anoenderana kuti vagadzirire kugadzirwa kwemamwe ma antibodies, kukurudzira kutsvagurudza kwesainzi, kugadzira maturusi ekuongorora kliniki, uye kuita kuti kuwanikwa nekugadzirwa kwemishonga mitsva kuitike. Makamera, alpacas, Liama, nedzimwe ngamera muCamelidae dzinogona kugadzira nanobody (single domain antibody) maraibhurari kuburikidza nekuunganidza ropa rekudzivirira muviri uye kubvisa maPBMC. Tsuro nedzimwe mhuka dzinogona kubayiwa majekiseni uye ma antibodies anogona kubviswa mumiviri yadzo. Mushure mekucheneswa, dzinogona kushandiswa kugadzira ma polyclonal antibodies. Mbeva, makonzo, nezvimwewo zvinogona kushandiswa kugadzira ma monoclonal antibodies nekubatanidza masero eB akabudiswa neimmune nemasero e myeloma. Pamberi pekubayiwa, kutariswa kunofanira kupihwa kune misimboti ye3R (kutsiva, kuderedza, kunatswa), izvo zvinogona kuderedza zvakanyanya huwandu hwemhuka dzinoshandiswa kusvika padanho rinodiwa, kuvandudza maitiro ekuedza, uye kuderedza kutambura kwemhuka. Dhizaini chaiyo yekuedza, kusarudzwa kwema antibodies akakodzera, uye maitiro akasimba ekusimbisa anofanirawo kuteverwa.

Kupatsanurwa kwema immunogens - kugadzirwa kwe nanobody

Mhando dzeImmunogens	Muenzaniso	Kugadzirira Immunogen	Zvinyorwa
Mapuroteni Immunogens	Ma enzyme, mapuroteni, chepfu dzebhakitiriya, nezvimwe zvinhu	Sarudza masisitimu akasiyana ekutaura mapuroteni: E. coli, mbiriso, masero ezvipembenene, masero emhuka dzinoyamwisa, uye sisitimu isina masero	Tarisa zvinhu zvakaita sekupisa, nguva, pH, uye zvinhu zvinokanganisa kugadzirwa kweprotein.
Nucleic Acid Immunogens	DNA, RNA	DNA Immunogen: Gadzira plasmid DNA, gadzira mavector akakodzera ekutaura kwemajini anotarisirwa, masero ekurima kuti awedzere plasmid DNA, uye kubvisa plasmid DNA yakachena kwazvo. RNA Immunogen: Kudiwa Kwekuwedzera Chimiro Chengowani uye Muswe wePolyA Kudzivirira Kuora	Pakugadzirira ma immunogen eDNA, kungwarira kunofanira kupiwa kumasero akakodzera emuviri, uye pakugadzirira ma RNA immunogens, kungwarira kunofanira kupiwa pakudzivirira kuparara kweRNA.
Mavhairasi ekudzivirira muviri	Jekiseni rekudzivirira hutachiona hwese, jekiseni rechikamu chidiki, jekiseni rekudzivirira hutachiona (adenovirus, lentivirus, nezvimwewo), jekiseni remRAN.	Pakugadzirira mushonga wekudzivirira hutachiona hwese usina kushanda, hutachiona hunotanga hwamiswa kuti pave nekuchengetedzeka. Kuongororwa kwechepfu kunoshandiswa kuona kana mushonga wacho wapera. Majekiseni ekudzivirira hutachiona anongoda mapuroteni epamusoro pehutachiona semaantigen uye anofanirwa kufunga nezvekushanda kwemuviri.	Zvinobatsira zvinofanira kuwedzerwa kuti zvipe simba rekudzivirira muviri, kutarisisa hutachiona hunofanira kuregedzwa kushanda, mazita akakodzera ehutachiona anodiwa kuti pave nesimba rekudzivirira muviri, uye kuchengetedzeka kwegadziriro yacho kunofanira kudzorwa zvakasimba.

Maitiro ekubaiwa nhomba mhuka kana nguva yekudzivirira

Kusarudzwa kweMhuka uye Kugadzirira Antigen

Kusarudzwa kwemhuka: Sarudzai mhuka dzine zera rakakodzera uye dzine muviri wakagwinya kubva mumapoka ane hutano hwakanaka, uye ongororai ropa uye ongororai zvirwere kuti muve nechokwadi chekuti hapana zvirwere zvinotapukira kana zvimwe zvinetso zvehutano.

Kugadzirira immune system: Immunogens ndiyo kiyi yekukonzera mhinduro dzemuviri uye inoda kusarudzwa kwemapuroteni akakodzera, mapeptide, kana zvimwe zvinhu zvakakodzera zvichienderana nezvinodiwa neantibody iri kutarisirwa.

Alpaca inogona kubaiwa majekiseni nemaantigen 1-3 panguva imwe chete, nehuwandu hweantigen hwe1-2mg pakubaiwa kwega kwega uye huwandu husingasviki 2mL. Pamberi pekubaiwa, antigen neadjuvant zvinosanganiswa muchikamu che1:1 kuti zvigadzire musanganiswa wakafanana, unochengetwa pa4 ℃.

Immunological alpaca

Nyora nhamba yenzeve yealpaca isina chinhu wotanga kuyedza kubaya. Isa jekiseni muma lymph nodes ari pedyo nemutsipa wealpaca kumativi ese, uine mapoinzi maviri kudivi rimwe nerimwe uye inenge 0.4mL ye emulsified antigen inopinzwa panzvimbo imwe neimwe. Mushure mekubaya, tarisai kwehafu yeawa kuti musimbise kuti alpaca haina zviratidzo zvekusagadzikana. Kubaya jekiseni kunofanira kuitwa mavhiki maviri ega ega, uye kubaya jekiseni kanenge ka4.

Kuunganidzwa kweropa

Mushure mekubaiwa majekiseni mana kwemazuva mashanu kusvika manomwe, torai 50ml yeropa kubva mutsinga yemutsipa yealpaca.

Kuparadzaniswa kweropa

Masampuli eropa anotorwa kuti aongororwe immune system isati yapihwa mushonga wekudzivirira maantigen, uye 5mL yeropa inotorwa nguva imwe neimwe; Pazuva rimwe chetero, ropa rakaiswa mu centrifuge pa 400 xg kwemaminitsi makumi matatu uchishandisa 25 ℃ centrifuge yakatonhodzwa. Chikamu chepamusoro che serum chakaparadzaniswa ndokuchengetwa kuti chiongororwe antibody titer inotevera.

Malymphocyte akasiyana

Kutanga wedzera 15mL yemushonga wekuparadzanisa masero, wobva wawedzera 15mL yeropa zvishoma nezvishoma muchubhu ye50mL centrifuge. Chenjerera uye nonoka paunenge uchiisa ropa kudzivirira kusanganiswa kweropa nemushonga wekuparadzanisa. Precooled centrifuge pa25 ℃, centrifuge pa400 xg kwemaminitsi makumi matatu, uye chengeta serum yepamusoro muchubhu itsva yecentrifuge pa -80 ℃; Shandisa pipette kuti ufukidze masero emuviri epakati akaita sedonje muchubhu itsva ye50ml centrifuge. Wedzera 10mL PBS buffer patembiricha yekamuri kuchubhu yega yega, centrifuge pa25 ℃ uye 400 xg kwemaminitsi makumi maviri. Bvisa supernatant, wedzera 5mL yePBS buffer patembiricha yekamuri kuchubhu yega yega, sanganisa zvinyoro nyoro, verenga huwandu hwemasero uchishandisa hemocytometer, wozo centrifuge pa25 ℃ uye 400 xg kwemaminitsi makumi maviri. Bvisa supernatant wobva wanyungudutsa ma lymphocytes anowanikwa neRNAiso Plus zvichienderana nehuwandu hwemasero kuti uwane 10 ^ 7/mL cell lysate, iyo inochengetwa pa -80 ℃.

kuwanikwa kwemasoja ekudzivirira chirwere

Mufananidzo 1: Kuratidzwa kweSchematic kweMaitiro Akasiyana eKuwana MaAntibody uye Matanho Ekuyedza Anobatanidzwa. (Mufananidzo wakabva: Laustsen, Andreas H. nevamwe.)

Vatengi vanopa kudzora kwakasimba kwemhando ye immunogens

Mhando dzeImmunogens	Chinodiwa	Nzira Yekudzora Hunhu
Mienzaniso yePeptide / Molecule Diki	Mamiriro ekunyungudika (zvikuru zvichienderana nekuti achamisa maphages here), chimiro chemamorekuru/peptide sequence, mushumo we synthesis (HPLC/MS/HNMR)	Kuonekwa kweHPLC/MS
Mienzaniso yeMapuroteni	SDS-PAGE/WB, mamiriro ekugadzirisazve, buffer, ruzivo rwezita, huwandu hwese (20-50ug), huwandu hwese, kuchena, nezvimwewo	SDS-PEJI/WB
Mienzaniso Yemasero	Rudzi rwesero (masero ekutanga/masero akagadziriswa majini), chimiro chesero (chitsva/chakanyungudutswa nechando), rudzi rwekukura (chinonamatira/chakamiswa), zvinodiwa zvekurima, huwandu hwemasero anopiwa, huwandu hwemasero anopiwa, masero akagadziriswa majini kuti ave nechokwadi chekuti zvinangwa zvekuongorora zvinoratidzwa zvakagadzikana (antibody inoenderana nevatengi), masero akanyungudutswa nechando kuti aone mwero wekupora	Kunyanya kutarisa masero akagadziriswa majini, uchishandisa ma antibodies ekutanga anopiwa nevatengi kuti vaone ELISA yekutaura chinangwa (kambani yedu inopa ma antibodies echipiri eHRP), kuona flow cytometry (kambani yedu inogona kupa ma antibodies echipiri e fluorescent).

Ndezvipi zvinoshandiswa pakudzivirira zvirwere zvemhuka?

+

Ma antibodies ePolyclonal anogona kugadzirwa nekubaiwa majekiseni ekudzivirira mhuka, ma antibodies emonoclonal anogona kugadzirwa nekubatanidza masero eB nemasero emyeloma, uye maPBMC anogona kutorwa kubva kumatsime ekamera (alpaca, ngamera, alpaca, nezvimwewo) kuti agadzire mafomu akasiyana-siyana e antibodies akadai se nanobodies, Fab fragments, scFv antibodies fragments, nezvimwewo.
Ndezvipi matanho ekudzivirira hutachiona hwemhuka?

+

Kuti pave nema nanoantibodies emhuka dzinobva pangamera, panodiwa majekiseni mashanu kusvika matanhatu. Samples dzeropa dzinofanira kutorwa senzira yekudzora dzisati dzabatwa, nechikamu chemavhiki maviri pakati pekudzivirira kwega kwega. Mushure mekudzivirira kwechina, ropa rakanaka uye ropa risina kubatwa rinofanirwa kutorwa kuti ELISA ione kuti titer yacho ndeyei. Kana titer iri pasi pe10 ^ 6, huwandu hwemajekiseni hunofanira kuwedzerwa kusvika titer yasvika padanho rakakodzera. Kune mamwe majekiseni emhuka akadai sekudzivirira tsuro (chiyero chakanaka kana chakajairika cheprotein antigen chekudzivirira tsuro i50-1000ug), kudzivirira mbeva (chiyero chakanaka kana chakajairika cheprotein antigen chekudzivirira mbeva i5-50ug, chinoda 10 ^ 6 masero kana iri sero, uye 10-50ug ye nucleic acid kana carbohydrates), kudzivirira mbudzi (chiyero chakanaka kana chakajairika cheprotein antigen chekudzivirira mbudzi i250-5000ug), nezvimwewo, kutarisirwa kunofanira kupihwa kune dosage, kuchengetedzeka, uye kushanda kwe immunogens.
Nzira yekusarudza mhuka dzinodzivirira zvirwere?

+

Zvichienderana nerudzi rweantibody inodiwa nemutengi, kana uchida kugadzira nanobodies kana zvimwe zvidimbu zveantibody, unogona kusarudza mhuka dzinobva pangamera (ngamera, alpacas, alpacas) kuti ubatwe. Kana uchida kugadzira monoclonal kana polyclonal antibodies, unofanirwa kusarudza mhando dzakakodzera zvichienderana nehuwandu hweserum, antigen, uye antigen source. Huwandu hwepamusoro hweserum yetsuro i500ml, iyo inoda huwandu hushoma hweantigen uye ndiyo sarudzo huru yekugadzira polyclonal antibodies. Huwandu hwepamusoro hweserum yembeva i2ml, uye huwandu hwepamusoro hweserum yemakonzo i20ml, iyo inowanzova sarudzo yakanakisisa ye monoclonal antibodies. Ma antibodies anogadzirwa nembudzi ane hukama hwakanyanya, uye Alpha Lifetech inogona kugadzirisa hurongwa hwekudzivirira hwakasiyana kwauri zvichienderana nezvaunoda.
Maantigen anofanira kushandiswa pamwe chete ne adjuvants here? Ungasarudza sei?

+

Kune mhando mbiri dzezvinobatsira: zvinobva mumafuta uye zvinobva mumvura. Kushandisa zvine mwero zvisingabatsiri kwakakosha pakukurudzira mhinduro dzakasimba dzemaantigen kune maantigen anonyungudika. Kuburitswa kwenguva refu kwezvinobatsira zvinoreva kuti maantigen mashoma anogona kushandiswa, uye mhinduro dzemaantibody dzinogara kwenguva refu. Jekiseni rekutanga rinofanira kushandiswa pamwe chete neadjuvant. Kazhinji zvinokurudzirwa kushandisa Freund's adjuvant kana huwandu hwe immunogen huri hushoma.
Nzira dzekudzivirira nadzo kubaya alpaca?

+

(1) Kusarudzwa kwealpaca neantigen yekudzivirira muviri ndiyo kiyi yekubudirira kwekudzivirira muviri. Kusarudza ngamera yakayerwa zvakanaka hazvikurudzirwi. Kuchena uye chimiro chakakodzera cheantigen dzekudzivirira muviri zvakakosha pakuongorora ma antibodies akakodzera kushandiswa mushure mekudzivirira nealpacas. Kuchena kweprotein antigens kazhinji hakupfuuri 90%.

(2) Kupatsanurwa kwemalymphocyte: Kupatsanurwa kwemasero panguva yakakodzera kunogona kudzivirira kubuda kweropa mushure mekuunganidzwa kweropa kuti pave nemhedzisiro yakanaka yekupatsanurwa.

(3) Kutenderera kwemuviri kunogona kukanganisa mhinduro yemuviri kuzvirwere: nguva yekudzivirira muviri kwevhiki imwe kusvika mbiri inobvumira alpacas kuti ive nemhinduro yakanaka yemuviri kuzvirwere zvakawanda.

adjuvant yakagadzirwa yakazvimiririra

Mushonga wedu wekubatsira muviri wakasiyana wakagadzirwa kuti ubatsire masoja emuviri ealpaca, zvichipa simba rakawanda uye hunhu hwakasiyana pane mishonga yechinyakare.

Kupihwa majekiseni ekudzivirira
mwero

Tinovimbisa kupedza kubaya majekiseni mukati menguva yakatarwa, kupa serum ine titer yakawanda, uye kunyora nhanho imwe neimwe kuburikidza nemishumo yakadzama.

Kudzora Hunhu hwepurojekiti

Chirongwa chega chega chinoitwa maitiro ekudzora mhando kuti chive nechokwadi chekuti masoja ekudzivirira chirwere anosangana nezvinodiwa zvepamusoro kune vatengi.

zvakajekaAlpacaKumashure

Tine vanhu vane hutano hwakanaka vanopa rutsigiro kumapurogiramu akasiyana-siyana ekudzivirira.

Kana muine mibvunzo, ndapota inzwa wakasununguka kutibata chero nguva.

Leave Your Message

Basa Rinonyanya Kuonekwa

0102