Utumiki Wopanga Ma Antibody a Monoclonal

Alpha Lifetech Inc.Zingapereke kuneneratu kwa epitope, kupanga peptide, kuwonetsa mapuloteni, kupanga ma antibodies a polyclonal, kapena monoclonal komanso kuyeretsa ma antibodies kuchokera ku chitukuko cha maselo, madzi a ascites kapena seramu yonse.

Alpha Lifetech Inc.ali ndi luso lopereka chithandizo chamtundu wa cell line kwa makasitomala osiyanasiyana kuphatikizapo ma cell a hybridoma, genome editing cell line, ma cell line ogwetsa, ndi ma cell line owonjezera mphamvu. Timayesetsa kupereka chithandizo chamtundu wa cell line kwa kasitomala aliyense ndipo ntchito zathu ndi zotsimikizika komanso zodalirika.

Njira Yopangira Ma Antibody

1. Kapangidwe ndi kukonzekera kwa antigen
Ma antigen awa akhoza kukhala mapuloteni, ma peptide, kapena mamolekyu ena. Kupanga ma immunogen kumafuna njira yokwanira yomwe imagwirizanitsa chidziwitso cha kapangidwe ka ma antigen, maphunziro a chitetezo chamthupi, ndi njira zoperekera. Alpha Lifetech ili ndi ukadaulo wopanga ma antigen womwe ungathe kupanga ma immunogen kwa makasitomala omwe akusowa thandizo.
Alpha Lifetech imapereka zinthu zopangira mapuloteni ophatikizana kuti makasitomala asankhe. Tili ndi njira zosiyanasiyana zopangira mapuloteni, monga njira yowonetsera E. coli, njira yowonetsera yisiti, njira yowonetsera tizilombo toyambitsa matenda a baculovirus, njira yowonetsera tizilombo toyambitsa matenda a mammary, ndi zina zotero, kuti tipange mapuloteni ophatikizana omwe mukufuna kuti mufufuze.

2. Katemera wa ziweto
Alpha Lifetech ikhoza kupereka ma immunogens kwa makasitomala, ndipo nyama zodzitetezera monga mbewa, akalulu, nkhosa, alpacas, nkhumba za Guinea, hamsters, ndi zina zotero zilipo kuti musankhe.

3. Njira zopangira ma antibodies a monoclonal

●Kupanga Mabs ndi ukadaulo wa Hybridoma
Hybridoma ndi selo losakanikirana lomwe limapangidwa chifukwa cha kusakanikirana kwa mitundu iwiri ya maselo, omwe ndi mzere wa maselo a mbewa a myeloma ndi maselo a plasma (lymphocyte B), momwe selo la heterozygous limatha kupanga ma antibodies osalekeza motsutsana ndi antigen yomwe imakonda mu vitro. Kupanga mzere wa maselo a hybridoma kumakhala ndi magawo atatu: kapangidwe ka antigen (ma haptens, ma molecule ang'onoang'ono, ndi ma peptide), katemera wa ziweto, kusakanikirana kwa maselo, ndi kuwunikira bwino kwa clone. Pogwiritsa ntchito njira zapadera zodzitetezera, timapeza mphamvu zapamwamba kwambiri zosakanikirana kwa maselo (ma hybridoma 1-10 pa maselo chikwi a B) ndi ma antibodies apamwamba, zomwe zimapangitsa kuti tipambane kwambiri pamayeso otsatira omwe amaphatikizapo kuwunikira kwa biochemical, kuyesa kwa maselo a in-vitro, ndi maphunziro a ziweto.

●Kupanga kwa Mabs pogwiritsa ntchito ukadaulo wowonetsera phage
Kuwonetsa kwa phage kumapereka njira ina yopangira ma antibodies a monoclonal. Ma B-lymphocyte amachotsedwa m'magazi a nyama ndipo mRNA yawo imachotsedwa. Kudzera mu PCR, mRNA iyi imasinthidwa kukhala cDNA, yomwe imakulitsa magawo onse a VH ndi VL. Magawo awa amapangidwa kukhala vector, nthawi zambiri ngati zidutswa zosinthika za unyolo umodzi (scFv), pamodzi ndi puloteni ya PIII ya bacteriophage. Pambuyo pake, kapangidwe kameneka kamagwiritsidwa ntchito kupatsira E. coli, zomwe zimapangitsa kuti pakhale laibulale yokhala ndi maselo pafupifupi 10^10 kudzera mu kubayidwa ndi phage yothandizira. E. coli imatha kutulutsa bacteriophage yomwe ili ndi magawo a VH ndi VL ngati gawo la khungu lake. Pambuyo pake, magawo enaake a VH ndi VL omwe akuyang'ana antigen yosangalatsa amatha kusankhidwa ndikugwiritsidwa ntchito kubwezeretsanso E. coli ndi bacteriophage. Maselo okhala ndi plasmid amachotsedwa ndikusanjidwa.

4. Kuwunika kwa fusion

●Kusakanikirana ndi Kusankha kwa Maselo
Maselo a B odulidwawo amaphatikizidwa ndi maselo a myeloma, omwe ndi maselo a khansa omwe amatha kufalikira kosatha mu ulimi. Kusakanikirana kumeneku kumachitika pogwiritsa ntchito njira monga polyethylene glycol (PEG) fusion.
Pambuyo posakanikirana, maselo a hybridoma amakula m'njira yosankha yomwe imalola ma hybridoma okhawo kuti akhale ndi moyo. Njirayi nthawi zambiri imakhala ndi aminopterin, yomwe imaletsa kukula kwa maselo a myeloma osasakanikirana.

●Kuwunika ndi Kuphimba
Maselo a hybridoma omwe amabwera chifukwa cha matendawa amafufuzidwa kuti aone ngati pali ma antibodies enieni a antigen yomwe ikufunidwa. Izi nthawi zambiri zimachitika pogwiritsa ntchito njira monga enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, ndi flow cytometry.

Maselo a hybridoma omwe amapanga antibody akapezeka, amapangidwa kuti apange maselo ofanana. Izi zimatsimikizira kuti antibody yomwe imapangidwa ndi selo lililonse la hybridoma ndi yofanana.

5. Kutsimikizira Ntchito ya Antibody
Alpha Lifetech imapereka chitsimikizo cha ntchito ya ma antibodies, monga Western blotting, immunohistochemistry, kapena functional assays, kuti iwonetse ma antibodies a monoclonal ndikutsimikizira kutsimikizika kwa ma antibodies, kukondana, ndi magwiridwe antchito.

6. Kukonza bwino ma antibodies
Kuyeretsa ma antibodies kuchokera ku supernatant ya culture pogwiritsa ntchito njira monga protein A kapena protein G affinity chromatography, ndi Alpha Lifetech zingaperekenso njira zosinthira ma antibodies kuti ziwongolere antibodies.

7. Kupanga ma antibodies
Alpha Lifetech imatha kuwunika ma antibodies oyenera kuti agwiritsidwe ntchito poyesa kuchuluka kwa ma antibodies komanso kupanga kwakukulu.

FAQFAQ

Mafunso Ofunsidwa Kawirikawiri Pakukula kwa Ma Antibody a Monoclonal

1

Kodi mungasankhe bwanji fusion hybridoma yoyenera?

Pa nthawi yosakaniza, mbale 10-15 za microtiter 96-well zimabzalidwa ndi chisakanizo cha fusion hybrid. Mbale iliyonse imadyetsedwa HAT-IMDM selection media ndikusungidwa mu carbon dioxide incubator pa madigiri 37 Celsius. Patatha masiku 9 - 14 kuchokera pamene fusion idapangidwa, supernatants yosakanikirana imayesedwa kuti ione ngati pali antibody yeniyeni yomwe ikufunika.
2

Kodi mungatani kuti muwongolere bwino kusankha?

Kuphatikizika kwa maselo a plasma ndi maselo ofanana nawo a myeloma sikugwira ntchito 100%. Ngakhale pakakhala zinthu zabwino komanso zolimbikitsa kwambiri, kuphatikizika kwa maselo kumapangabe kusakaniza kwa maselo osalumikizana ndi olumikizana omwe amafunika kulekanitsidwa. Kuti njira yosankha ikhale yogwira ntchito bwino, maselo a myeloma omwe amagwiritsidwa ntchito pophatikizika alibe HGPRT, enzyme yofunika kwambiri mu njira yopulumutsira nucleotide. Kenako kusakanizako kunakulitsidwa mu HAT medium, komwe maselo okhawo omwe ali ndi enzyme ya HGPRT, ma hybridoma omwe adalandira HGPRT kuchokera ku maselo a plasma, ndi omwe anali ndi mphamvu.
3

Kodi mungayang'ane bwanji mizere ya maselo a hybridoma kuti muwone ngati pali ntchito ya antibody?

Kuwunika mitundu yosiyanasiyana ndi gawo lofunika kwambiri. Nthawi zambiri, hybridoma, clone, ndi subclone supernatant screening zimaperekedwa kudzera mu enzyme-linked immunosorbent assay (ELISA), Western blot assay, ndi fluorescence activated cell sorting (FACS). Tidzasankha kuchita kuwunika konse kwa supernatant mu labotale yathu.
4

Perekani malangizo atsatanetsatane a momwe mungayezerere ntchito ya antibody?

Ma hybridoma supernates omwe akuyenera kuyesedwa akhoza kukhala pakati pa zitsanzo 500 mpaka 1,440 ndipo adzakhala okonzeka kuyesedwa pofika masiku 9-14. Tikhoza kuonekera mkati mwa masiku atatu kapena asanu kapena tonse tingakhale okonzeka tsiku lomwelo, choncho ndikofunikira kuti labotale ya kasitomala ikhale yokonzeka milungu ingapo pasadakhale ndi mayeso enaake oyesera achiwiri omwe mungasankhe.
5

Chifukwa chiyani ma hybridomas abwino ayenera kugawidwa m'magulu awiri?

Pambuyo poti ma Wells omwe ali ndi kachilombo adapezeka panthawi yoyesa koyamba kwa ELISA, ma hybridomas adasamutsidwira ku voliyumu yayikulu, mwachitsanzo, mbale 24 za zitsime, pokonzekera njira yogwiritsira ntchito subcloning. Subcloning ya ma hybridomas nthawi zambiri imachitika pogwiritsa ntchito njira yochepetsera kufalikira kwa ma monoclones okhazikika kuti atsimikizire kuti ma monoclones okhazikika akupezeka. Njirayi imaphatikizapo kuchulukitsa kukula kwa ma hybridomas ndikuwagawa m'ma mbale 96 a zitsime kuti akwaniritse monocloniality (selo limodzi pa chitsime). Kuchepetsa kufalikira kuwiri kuyenera kuchitika kuti achepetse chiopsezo chokhala ndi ma hybridoma osakanikirana.
6

Kodi ubwino wogwiritsa ntchito ukadaulo wa hybridoma pofufuza ma antibodies ndi wotani?

Maselo osakanikirana atamandidwa chifukwa cha luso lawo lopanga ma antibodies okhala ndi mphamvu zambiri, kukhazikika, komanso kulunjika mwanjira yotsika mtengo kwambiri.