ENHANCEMENT OF ELISAS FOR SCREENING PEPTIDES IN EPITOPE PHAGE DISPLAYLIBRARIES

The complete analysis of epitope phage display libraries requires sens itive assays capable of detecting peptides expressed on phage that hav e a wide range of affinities for antibody. We have compared two ELISAs , a 'direct' assay where the phage is captured by an anti-phage antibo dy and the peptide detected by the antibody used for screening, and a 'reverse' assay where the antibody used for screening is first coated on the well and the binding of phage detected by the anti-phage antibo dy. We demonstrate, by comparing two fUSE5 derived phage bearing five peptides reacting with the anti-cryptococcal polysaccharide antibody 2 H1, that the reverse ELISA is the more sensitive assay. Further, there is a limit in affinity, here around 1 mu M, above which phage clones are negative by the direct ELISA. We describe an enhancement of the di rect assay by mixing 2H1 with 3-fold excess of anti-heavy or anti-ligh t chain antibody. The resulting polymerization of 2H1 induces an incre ase in antibody avidity that is responsible for the enhancement. The e nhanced direct ELISA allowed rapid and sensitive detection of positive clones and is easily inhibited by free peptide, while the reverse ELI SA is not. The enhanced ELISA has also been used successfully for immu nological screening of intermediate libraries, allowing detection of r are positive clones that would otherwise be lost. The combination of t he three ELISAs, reverse, direct, and enhanced direct, should provide a way to rank phage clones into three classes: very low, low, and high affinity, providing information previously obtained only by the synth esis and testing of many peptides.