Basa rekugadzira maAntibody eMonoclonal

Alpha Lifetech Inc.inogona kupa kufanotaura kwe epitope, kugadzirwa kwe peptide, kuratidzwa kweprotein, kugadzirwa kwe polyclonal, kana monoclonal antibody pamwe nekucheneswa kwe antibody kubva mukumera kwemasero, ascites fluid kana serum yese.

Alpha Lifetech Inc.ine ruzivo mukupa vatengi masevhisi akasiyana-siyana emasero akasimba anosanganisira masero ehybridoma, mitsetse yemasero ekugadzirisa genome, mitsetse yemasero ekudonhedza pasi, uye mitsetse yemasero ekuwedzera. Tinoedza kupa masevhisi emhando yepamusoro kune mutengi wese uye masevhisi edu akasimbiswa uye akavimbika.

Nzira yekuvandudza maAntibody

1. Kugadzira uye kugadzirira maAntigen
Maantigen aya anogona kuva mapuroteni, mapeptide, kana mamwe mamorekuru. Kugadzira ma immunogens kunoda nzira yakazara inobatanidza ruzivo rwemaumbirwo eantigen, immunology, uye masisitimu ekutumira. Alpha Lifetech ine tekinoroji yekugadzira maantigen inogona kugadzira ma immunogens kune vatengi vanoshaya.
Alpha Lifetech inopa zvigadzirwa zveprotein zvinosanganiswa kuti vatengi vasarudze kubva. Tine nzira dzakasiyana-siyana dzekutaura mapuroteni, dzakadai seE. coli expression system, yeast expression system, baculovirus-insect expression system, mammalia expression system, nezvimwewo, kuti tigadzire mapuroteni anosanganiswa aunoda kuti titsvage.

2. Kudzivirira mhuka
Alpha Lifetech inogona kupa mishonga yekudzivirira muviri kuvatengi, uye mhuka dzinodzivirira muviri dzakadai sembeva, tsuro, makwai, alpacas, Guinea pigs, hamsters, nezvimwewo zviripo kuti usarudze kubva.

3. Nzira dzekugadzira maantibody emonoclonal

●Kugadzirwa kweMabs netekinoroji yeHybridoma
Hybridoma iboka remasero akagadzirwa nekubatanidzwa kwemarudzi maviri emasero, kureva masero embeva yemyeloma nemasero eplasma (lymphocyte B), umo masero eheterozygous anogona kugadzira ma antibodies anoenderera mberi achirwisa antigen inodiwa mu vitro. Kugadzirwa kweboka remasero ehybridoma kune zvikamu zvitatu: dhizaini yeantigen (haptens, mamorekuru madiki, uye mapeptide), kubaiwa mhuka, kubatanidzwa kwemasero, uye kuongorora kwakanaka kwe clone. Tichishandisa nzira dzakasiyana dzekudzivirira, tinowana kushanda kwakanyanya kwemasero-kusanganiswa (1-10 hybridomas pamasero eB chiuru) uye ma antibody titers akakwira, zvichiwedzera mwero wekubudirira kwedu mumiedzo inotevera inosanganisira kuongorora biochemical, in-vitro cell-based assays, uye zvidzidzo zvemhuka.

●Kugadzirwa kweMabs netekinoroji yekuratidza ma phage
Kuratidzwa kwePhage kunopa imwe nzira yekugadzira ma antibodies e monoclonal. B-lymphocytes dzinobviswa muropa remhuka uye mRNA yavo inobviswa. Kuburikidza nePCR, mRNA iyi inoshandurwa kuita cDNA, iyo inowedzera zvikamu zvese zveVH neVL. Zvikamu izvi zvinobva zvaunganidzwa kuita vector, kazhinji se single-chain variable fragments (scFv), pamwe chete nePIII protein yebacteriophage. Zvadaro, chimiro ichi chinoshandiswa kutapurira E. coli, zvichikonzera kugadzirwa kweraibhurari ine masero angangoita 10^10 kuburikidza nekubaiwa ne helper phage. E. coli inokwanisa kuburitsa bacteriophage ine zvikamu zveVH neVL sechikamu che coat yayo. Mushure meizvi, zvikamu zveVH neVL zvakanangana ne antigen zvinokwanisa kusarudzwa uye kushandiswa kubatanidza E. coli ne bacteriophage. Masero ane plasmid anobva aparadzaniswa uye arongwa.

4. Kuongororwa kwekubatana

●Kusanganiswa Kwemasero Uye Kusarudzwa
Masero eB akakohwewa anobva asanganiswa nemasero emyeloma, ayo ari masero ekenza anogona kukura nekusingaperi mukukura. Kusanganiswa uku kunowanzoitwa uchishandisa nzira yakaita se polyethylene glycol (PEG) fusion.
Mushure mekusanganiswa, masero ehybridoma anorimwa nenzira yakasarudzwa inobvumira mahybridoma chete kuti ararame. Nzira yacho inowanzova neaminopterin, iyo inodzivirira kukura kwemasero emyeloma asina kusanganiswa.

●Kuongorora uye Kusunga
Masero ehybridoma anobuda anoongororwa kuti aone kana paine ma antibodies chaiwo kune target antigen. Izvi zvinowanzoitwa uchishandisa nzira dzakadai se enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, uye flow cytometry.

Kana masero ehybridoma anogadzira antibody aonekwa, anoumbwa kuti agadzire huwandu hwemasero akafanana. Izvi zvinoita kuti antibody inogadzirwa nesero rega rega rehybridoma ive yakafanana.

5. Kusimbiswa kweKushanda kweAntibody
Alpha Lifetech inopa humbowo hwekuti ma antibodies anoshanda sei, senge Western blotting, immunohistochemistry, kana functional assays, kuti ione ma antibodies e monoclonal uye isimbise kuti ma antibodies acho akasiyana sei, affinity, uye mashandiro awo.

6. Kugadzirisa maAntibody
Kucheneswa kwema antibodies kubva ku culture supernatant uchishandisa nzira dzakadai se protein A kana protein G affinity chromatography, uye Alpha Lifetech inogonawo kupa nzira dzekugadzirisa ma antibodies kuti ivandudze antibody affinity.

7. Kugadzirwa kweAntibody
Alpha Lifetech inogona kuongorora ma antibodies anodiwa kuti aonekwe kuti anoshandiswa sei pakuongorora huwandu hwemasero uye kugadzirwa kwakawanda.

mibvunzo inowanzo bvunzwamibvunzo inowanzo bvunzwa

Mibvunzo Inowanzo bvunzwa nezvekugadzirwa kwemasoja ekudzivirira chirwere emonoclonal

1

Nzira yekusarudza fusion hybridoma yakakodzera sei?

Munguva yekusanganiswa, mapurati e10-15 e96-well microtiter anodyarwa nemusanganiswa wefusion hybrid. Purati yega yega inopihwa HAT-IMDM selection media uye inochengetwa mu carbon dioxide incubator pa37 degrees Celsius. Mazuva 9 - 14 mushure mekusanganiswa, ma hybrid supernatants anoedzwa kuti aone kana paine antibody chaiyo inodiwa.
2

Nzira yekuvandudza sei kushanda zvakanaka kwesarudzo?

Kusanganiswa kwemasero eplasma nemasero e myeloma hakusi kushanda zvizere. Kunyangwe mumamiriro ezvinhu akanaka uye kukurudzira kunobudirira, kusanganiswa kwemasero kuchiri kugadzira musanganiswa wemasero asina kusanganiswa nemasero akabatana anofanira kuparadzaniswa. Kuti masero e myeloma anoshandiswa pakusanganiswa haana HGPRT, iyo enzyme inokosha munzira yekuponesa nucleotide. Musanganiswa wacho wakazogadzirwa mune imwe HAT medium, uko masero ane HGPRT enzyme chete, ma hybridomas akagara nhaka HGPRT kubva kumasero eplasma, ndiwo aishanda.
3

Nzira yekuongorora mitsetse yemasero ehybridoma kuti uone kana paine basa rema antibodies?

Kuongororwa kwemhando dzakasiyana dzemahybrids idanho rakakosha. Kazhinji, kuongorora supernatant yehybridoma, clone, uye subclone kunoitwa kuburikidza ne enzyme-linked immunosorbent assay (ELISA), Western blot assay, uye fluorescence activated cell sorting (FACS). Tichasarudza kuita kuongorora kwese kwe supernatant murabhoritari yedu.
4

Kupa mirayiridzo yakadzama yekuti ungaongorora sei mashandiro emasoja emuviri?

MaHybridoma supernates anofanira kuongororwa anogona kuva pakati pe500 kusvika 1,440 samples uye achava akagadzirira kuongororwa panosvika mazuva 9-14. Tinogona kubuda mukati memazuva matatu kusvika mashanu kana kuti tese tingave takagadzirira pazuva rimwe chete, saka zvakakosha kuti rabhoritari yevatengi igadzirire mavhiki pachine nguva nekuongorora kwechipiri kwavanoda.
5

Sei ma hybridomas akanaka achifanira kuverengerwa pasi pemagene?

Mushure mekunge Wells dzawanikwa panguva yekutanga kuongororwa kweELISA, mahybridomas akaendeswa kune huwandu hwakakura, kureva, maplate emvura makumi maviri nemana, mukugadzirira maitiro ekudzivirira kuumbwa kwemasero. Kuumbwa kwemasero emvura kunowanzoitwa nenzira yekuderedzwa kwemasero kuti ive nechokwadi chekuti mamonoclones akagadzikana abviswa. Nzira iyi inosanganisira kuparadzanisa zvirimwa zvemahybridoma uye kuaparadzira mumaplate emvura makumi mapfumbamwe nematanhatu kuti pave nemonoclonality (sero rimwe chete pachitubu). Kuderedzwa kwemasero maviri kunofanirwa kuitwa kuderedza njodzi yekuva nehuwandu hwemahybridomas akasanganiswa.
6

Ndezvipi zvakanakira kushandisa tekinoroji yehybridoma pakutsvaga ma antibodies?

Mitsetse yemasero ehybrid yakarumbidzwa nekukwanisa kwayo kugadzira masoja emuviri ane hukama hwakakura, kugadzikana, uye hunyanzvi nenzira inodhura zvishoma.