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PEDS Advance Access originally published online on February 4, 2009
Protein Engineering Design and Selection 2009 22(4):273-280; doi:10.1093/protein/gzp002
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Immunological applications of single-domain llama recombinant antibodies isolated from a naïve library

Ana Monegal1, Diletta Ami1, Chiara Martinelli1,3, He Huang2, Marisa Aliprandi1, Paola Capasso1, Chiara Francavilla3, Giuseppe Ossolengo1 and Ario de Marco1,4

1Consortium for Genomic Technologies, Biochemistry Unit, IFOM-IEO Campus, Via Adamello 16, 20139 Milano, Italy 2Department of Biological Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P.R. China 3 SEMM, IFOM-IEO Campus, Via Adamello 16, 20139 Milano, Italy

4 To whom correspondence should be addressed. E-mail: ario.demarco{at}ifom-ieo-campus.it

We describe the rapid isolation of single-domain recombinant antibodies in VHH format from a pre-immune llama library created in our laboratory. Such naïve library has demonstrated to be a versatile tool and enabled the isolation of several different antibodies for any of the six proteins panned in parallel. The binders specific for human fibroblast growth factor receptor 1 (FGFR1) were successively analyzed in more detail and resulted suitable for both western blot and immunofluorescence analyses. Several milligrams per liter of antibodies were purified by affinity chromatography and used for kinetic and thermodynamic characterization. Their KD was in the nanomolar range and they apparently bound a FGF receptor 1 domain not overlapping the region recognized by its physiological ligand FGF. Altogether, the collected data indicate that the new library can enable the recovery of binders of high affinity, specificity and functionality in the conventional immunological tests, avoiding the necessity of further maturation steps. Such results confirmed recent reports of high affinity pre-immune IgNARs and supported the choice of using large single-domain recombinant antibody naïve libraries as an alternative to the preparation of immune libraries for selecting monoclonal antibodies, at convenient cost and time conditions.

Keywords: FGFR1/immunofluorescence/llama/phage display/single-domain recombinant antibodies

Received August 4, 2008; revised December 19, 2008; accepted January 5, 2009.


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