MAPPING OF LINEAR EPITOPES RECOGNIZED BY MONOCLONAL-ANTIBODIES WITH GENE-FRAGMENT PHAGE DISPLAY LIBRARIES

Epitope mapping with mono- or polyclonal antibodies has so far been do ne either by dissecting the antigens into overlapping polypeptides in the form of recombinantly expressed fusion proteins, or by synthesizin g overlapping short peptides, or by a combination of both methods. Her e, we report an alternative method which involves the generation of ra ndom gene fragments of approximately 50-200 bp in length and cloning t hese into the 5' terminus of the protein III gene of fd phages. Select ion for phages that bind a given monoclonal antibody and sequencing th e DNA inserts of immunopositive phages yields derived amino acid seque nces containing the desired epitope. A monoclonal antibody (mAb 215) d irected against the largest subunit of Drosophila RNA polymerase II (R PB215) was used to map the corresponding epitope in a fUSE5 phage disp lay library made of random DNA fragments from plasmid DNA containing t he entire gene. After a single round of panning with this phage librar y, bacterial colonies were obtained which produced fd phages displayin g the mAb 215 epitope. Sequencing of single-stranded phage DNA from a number of positive colonies (recognized by the antibody on colony immu noblots) resulted in overlapping sequences all containing the 15mer ep itope determined by mapping with synthetic peptides. Similarly, we hav e localized the epitopes recognized by a mouse monoclonal antibody dir ected against the human p53 protein, and by a mouse monoclonal antibod y directed against the human cytokeratin 19 protein. Identification of positive colonies after the panning: procedure depends on the detecti on system used (colony immunoblot or ELISA) and there appear to be som e restrictions to the use of linker-encoded amino acids for optimal pr esentation of epitopes. A comparison with epitope mapping by synthetic peptides shows that the phage display method allows one to map linear epitopes down to a size only slightly larger than the true epitope. I n general, our phage display method is faster, easier, and cheaper tha n the construction of overlapping fusion proteins or the use of synthe tic peptides, especially in cases where the antigen is a large polypep tide such as the 215 kDa subunit of eukaryotic RNA polymerase II.