Research paper Validation of a stable recombinant antibodies repertoire for the direct selection of functional intracellular reagents Maria Elena Villani, Mariasole Di Carli, Marcello Donini, Giorgio Traversini, Chiara Lico, Rosella Franconi, Eugenio Benvenuto ⁎ , Angiola Desiderio ⁎ ENEA, Casaccia Research Centre, Dipartimento BIOTEC, Sezione Genetica e Genomica Vegetale, P.O. Box 2400 I-00100 Rome, Italy Received 26 February 2007; received in revised form 28 August 2007; accepted 5 September 2007 Available online 1 October 2007 Abstract We have previously generated a semi-synthetic single-chain variable fragments (scFv) phage display library built on a thermodynamically stable single-framework scaffold. All scFv antibodies selected from this repertoire showed high thermodynamic stability and were expressed as soluble molecules in bacterial cytoplasm. In this work, two complementary methodologies have been adopted to assess the functionality of library-derived scFvs as intracellular antibodies and to verify the possibility to directly use this repertoire for the selection of antibodies able to function in a reducing environment. The possibility to improve the performance of this highly stable antibody repertoire was evaluated subjecting the library to thermal denaturation and renaturation in the presence of a reducing agent before biopanning procedure. The scFv clones obtained after this treatment resulted the same isolated using standard biopanning conditions, suggesting that the selection efficiency of this repertoire is not affected by disulphide bonds formation. This evidence was confirmed by surface plasmon resonance analysis, measuring antigen affinity of a panel of library-derived scFv fragments both in oxidizing and reducing conditions. We observed perfectly comparable rate constants for antigen–scFv interactions in both antibody redox formats, demonstrating complete functionality also in the absence of intra-domain disulphide bonds. The experimental data point out that it is possible to straightforwardly isolate from this library scFvs with different specificities able to be functionally expressed in the cell cytoplasm. Hence, this library represents a valuable source of intrabodies for therapeutic applications. © 2007 Elsevier B.V. All rights reserved. Keywords: Phage display; scFv; Intrabody; Surface plasmon resonance 1. Introduction Antibodies represent ideal reagents for a wide range of applications due to their capacity to bind molecules with high affinity and specificity. The recombinant antibody technology allowed to engineer the complex structure of an immunoglobulin in different formats, the most effective being the single-chain Fv (scFv). In this format the heavy and light chain antibody variable domains (V H and V L respectively) are connected by a flexible linker, resulting in a smaller molecule that can be easily Journal of Immunological Methods 329 (2008) 11 – 20 www.elsevier.com/locate/jim Abbreviations: scFv, single-chain Fv antibody fragment; CDR, complementarity determining region; V H , heavy chain of an antibody Fv fragment; V L , light chain of an antibody Fv fragment; PVX, potato virus X; DTT, dithiotreitol; IPTG, isopropyl-β-D-galactopyranoside; PBS, phos- phate buffer saline; GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay; CMV, cucumber mosaic virus; HEL, hen egg lysozyme; redox, oxidation-reduction; HRP, horseradish peroxidase. ⁎ Corresponding authors. Benvenuto is to be contacted at Tel.: +39 06 30486347; fax: +39 06 30486545. Desiderio is to be contacted at Tel.: +39 06 30484176; fax: +39 06 30484808. E-mail addresses: benvenutoe@casaccia.enea.it (E. Benvenuto), desiderio@casaccia.enea.it (A. Desiderio). 0022-1759/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2007.09.003