Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 5.02

ISOLATION OF SINGLE-CHAIN Fv ANTIBODY FRAGMENTS AGAINST POTATO VIRUS X (PVX) FROM PHAGE DISPLAY LIBRARIES

M. DI CARLI, M.E. VILLANI, C. LICO, E. BENVENUTO

ENEA, BIOTEC-GEN, CR Casaccia, Via Anguillarese 301, 00060 Roma

PVX, scFv, phage display,molecular repertoires

In phage-display libraries, peptides or proteins are expressed as a fusion with the coat protein of the phage allowing a rapid selection of molecules with desired properties by a series of recursive cycles of selection. One of the most important application of phage-display has been the easy isolation of recombinant antibodies with desired specificity; for many application, one of the most versatile antibody format is the single-chain antibody (scFv) constituted by the V_H and V_L of the two variable domain fragments (scFv). This antibody format is a valuable tool for immunotherapy in plants (Benvenuto & Tavladoraki 1995) or diagnosis.

We isolated scFv antibodies specific to potato virus X (PVX) using two different phage display libraries. One, composed of intrinsically stable scFvs with a repertoire of approximately 5x 10⁷ different clones (the "F8 library" ) (Desiderio et al. 2001), and the other a human germline derived library composed of 6x10⁸ different clones (the "ETH2 library") (Pini et al. 1998).

After four rounds of selection and enrichment ("biopanning") on the immobilised virus, a panel of different phages clones were obtained from each library and individual clones were selected and tested for binding to the virus by enzyme-linked immunosorbent assay (ELISA). Approximately 65% of the clones from the "F8 library" were positive for the PVX while from the "ETH2 library".only 10% of the clones were positive. The clones that showed the highest binding to the virus were chosen for sequencing and further biochemical characterization. These scFvs fragments could represent a valuable tool for inexpensive immunodiagnosis, and once expressed functionally in transgenic plants can also be used for immunomodulation of PVX infection.

References

Benvenuto & Tavladoraki (1995) Trends Microbiol.3(7):272-5.

Desiderio et al. (2001) J Mol Biol. 13;310(3):603-15.

Pini et al. (1998) J Biol Chem 273(34):21769-76.