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Author:Arola, Henri
Title:Development of novel antibodies by phage display technology
Uusien vasta-aineiden kehittäminen faaginäyttö-teknologialla
Publication type:Master's thesis
Publication year:2011
Pages:iii + 115 + [9]      Language:   eng
Department/School:Kemian laitos
Main subject:Biokemia ja mikrobiologia   (Kem-30)
Supervisor:Nordström, Katrina
Instructor:Nevanen, Tarja
OEVS:
Electronic archive copy is available via Aalto Thesis Database.
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Location:P1 Ark Aalto  2031   | Archive
Keywords:phage display
antibody
Fab
hapten
faaginäyttö
vasta-aine
hapteeni
Abstract (eng): In the present work, novel antibodies against four small and challenging molecules (haptens) were developed by construction, selection and screening of an antibody library.
The haptens used in immunization were two thyroid hormones triiodothyronine (T3) and thyroxine (T4) and two catecholamine metabolites homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA).

The antibody library was constructed by cloning the heavy and light chain regions from mouse IgG genes sequentially into a phagemid vector.
The functional library size was 108.
These Fab fragments of antibodies were displayed on filamentous bacteriophages and specific antibodies were selected in a high-throughput format against the four antigens in parallel.
The selected clones were screened by using an ELISA test in automated robotic station.
The clones from the primary screening were further characterized by secondary ELISA.

The most promising clones were sequenced to see their diversity.
Altogether 15 different anti-T3 clones, 18 anti-T4 clones, 4 anti-HVA clones and 16 anti-5-HIAA clones were obtained, indicating that the used high throughput screening method from a multi-immunized library is a robust method and widely applicable for developing binders against various small molecules.
In addition, the multi-immunization saves animals, time and costs.
If needed, the multiple antibody clones provide excellent material for improving their properties by mutagenesis.
Abstract (fin): Työssä kehitettiin uusia rekombinanttivasta-aineita haasteellisille pienimolekyyleille (hapteeneille) rakentamalla vasta-ainekirjasto, josta seulottiin spesifisiä sitoutujia.
Immunisoinnissa käytetyt hapteenit olivat trijodotyroniini (T3) ja tyroksiini (T4), jotka ovat kilpirauhashormoneja sekä homovanilliinihappo (HVA) ja hydroksi-indolyyliasetaatti (5-HIAA), jotka ovat katekolamiinimetaboliitteja.

Vasta-ainekirjasto rakennettiin monistamalla hiiren IgG geeneistä erikseen raskas- ja kevytketjut ja yhdistämällä ne samaan vektoriin Fab-fragmenteiksi.
Valmiin vasta-ainekirjaston koko oli 108 erilaista kloonia.
Fab-fragmentit ilmennettiin filamentti-bakteriofagien pinnalla, jonka jälkeen spesifisiä vasta-aineita tehoseulottiin samanaikaisesti kaikille neljälle hapteenille käyttämällä automaattista magneettihelmien käsittelijää.
Eristetyt kloonit analysoitiin ELlSA-testillä automatisoidun robottiaseman avulla ja lupaavimmat kloonit sekvensoitiin niiden diversiteetin selvittämiseksi.

Vasta-ainekirjastosta saatiin eristettyä yhteensä 15 eri anti-T3 kloonia, 18 anti-T4 kloonia, 4 anti-HVA kloonia ja 16 anti-5-HIAA kloonia.
Tämä osoittaa, että multi-immunisoitu vasta-ainekirjasto ja tehoseulonta tuottavat tehokkaasti vasta-aineita monenlaisille pienmolekyyleille.
Lisäksi multi-immunisointi vähentää koe-eläinten tarvetta, työmäärää ja kuluja.
Tarvittaessa lukuisat työssä eristetyt vasta-ainekloonit tarjoavat erinomaisen lähtökohdan niiden ominaisuuksien parantamiseksi mutageneesilla.
ED:2011-12-15
INSSI record number: 43259
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