Abstract
(Englisch)
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In the first year of activity of the PEPSAC-MIMIC project, we have cloned two very large phage-display libraries of mutants of small globular proteins [the '434 library' and the 'PED-B' library; F. Viti, P. Pedrioli, B. Mitta, D. Neri (2001). 'Phage display libraries as a source of tumour-targeting agents'. Chimia, 55, 206-211].
In the second year of activity, our main activities have been:
a) The optimization of the display of libraries of small globular proteins on filamentous phage using hyperphage technology (Rondot S, Koch J, Breitling F, Dubel S. (2001) 'A helper phage to improve single-chain antibody presentation in phage display'. Nature Biotechnol. 19, 75-78). Evidence obtained in our lab and in other laboratories suggests that the efficiency of protein display on filamentous phage dramatically influences the performance of library selections.
b) The selection of the 434 and PED-B library with model antigens (globular proteins) and monoclonal antibodies specific for capsular antigens, provided by members of the PEPSAC-MIMIC Consortium.
We have performed selections with the two phage libraries mentioned above, against monoclonal antibodies specific for Vibrio cholera and Brucella.
The selections have been performed using submicromolar quantities of the monoclonal antibodies, followed by a biotinylated secondary antibody, followed by phage capture using streptavidin coated magnetic beads.
We have been able to isolate specific binding proteins. However, these proteins recognise the biotinylated secondary antibody, and not the primary antibody.
We hope to be able to overcome these problems, by using secondary biotinylated antibodies of different origin (e.g., rabbit and goat) in an alternated fashion, during the different rounds of panning.
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