Characterization of single chain antibody targets through yeast two hybrid. - Inserm - Institut national de la santé et de la recherche médicale Accéder directement au contenu
Article Dans Une Revue BMC Biotechnology Année : 2010

Characterization of single chain antibody targets through yeast two hybrid.

Résumé

BACKGROUND: Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain. RESULTS: We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region.Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. CONCLUSIONS: Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine.
Fichier principal
Vignette du fichier
1472-6750-10-59.pdf (1.14 Mo) Télécharger le fichier
1472-6750-10-59-S1.PDF (42.33 Ko) Télécharger le fichier
1472-6750-10-59-S2.PDF (43.98 Ko) Télécharger le fichier
1472-6750-10-59-S3.PDF (70.16 Ko) Télécharger le fichier
1472-6750-10-59-S4.PDF (52.72 Ko) Télécharger le fichier
1472-6750-10-59-S5.PDF (98.37 Ko) Télécharger le fichier
1472-6750-10-59.xml (73.27 Ko) Télécharger le fichier
Origine : Fichiers éditeurs autorisés sur une archive ouverte
Format : Autre
Format : Autre
Format : Autre
Format : Autre
Format : Autre
Format : Autre

Dates et versions

inserm-00617227 , version 1 (26-08-2011)

Identifiants

Citer

Ole Vielemeyer, Clément Nizak, Ana Joaquina Jimenez, Arnaud Echard, Bruno Goud, et al.. Characterization of single chain antibody targets through yeast two hybrid.. BMC Biotechnology, 2010, 10 (1), pp.59. ⟨10.1186/1472-6750-10-59⟩. ⟨inserm-00617227⟩
500 Consultations
423 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More