International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

E1. Using Phage Display in Functional Genomics

Peter Pavlik1, Rob Segal1, Daniele Sblattero2, Vittorio Verzillo2, Jianlong Lou2, Roberto Marzari3, Jim Marks4, Andrew Bradbury1,2.
1Los Alamos National Laboratory, Los Alamos, New Mexico
2International School for Advanced Studies, Trieste, Italy
3University of Trieste, Trieste, Italy
4University of California, San Francisco, San Francisco, California

We have developed novel recombinatorial methods for making phage antibody libraries (Nature Biotech 2000, 18, 75) which result in a final diversity of over 1e11 different phage antibodies. We have used this library, and others, in an attempt to derive a general method to select antibodies against gene products starting from sequence information.

As a first step towards this goal, selection of antibodies against recombinant proteins has been reduced to the microtitre format. A comparison of the antibodies selected using this protocol with the standard selection procedure shows that the antibodies selected are on the whole different, although there is some overlap. When gene products are available this is a very efficient way to select antibodies in a high throughput format.

Since gene products are often not available, selection on peptide surrogates of gene products has also been attempted. 90 scanning peptides corresponding to overlapping parts of four different proteins have been synthesised on microtitre pins and used to select phage antibodies. Some of the selected antibodies are able to recognise the full length protein. An analysis of the peptides which select antibodies recognising the full length protein has allowed us to develop an algorithm to predict which peptides are more likely to select useful proteins. We have used this algorithm to select antibodies against a number of different peptides derived from proteins, and many of the polyclonal and monoclonal antibodies selected recognise the full length protein from which they were derived.

Antibodies derived using phage display can be employed in the same way as traditional antibodies, including immunoprecipitation, immunoblotting, elisa, and immunofluorescence. As such, they can play essential roles in assigning gene function, including the characterization of spatiotemporal patterns of protein expression and the elucidation of protein-protein interactions.

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