Generation and epitope mapping of high-affinity scFv to eukaryotic elongation factor 1A by dual application of phage display

To generate specific tools for, in particular, localization studies of the eukaryotic elongation factor 1A (eEF1A), we have applied phage display in v arious formats to affinity-improve and map epitopes of two previously isola ted, low-affinity single-chain Fv (scFv) G3 and D1. The scFv differ in thei r reactivity toward the eEF1A isoforms, eEF1A-1 and eEF1A-2. By PCR-based r andomization of six residues within the variable light chain CDR3 (LCDR3), and subsequent phage-based affinity-selection, two 'families' of affinity-i mproved scFv were obtained. The scFv of highest affinity, A8, has a K-d of 9 nm to eEF1A-1. Interestingly, two affinity-improved scFvs have abnormally short LCDR3 consisting of two and four residues compared to 11 in the pare ntal scFv. Hence, the LCDR3 of the parental clones may play a modulating ra ther than a direct role in antigen-binding. Despite different preferences f or the eEF1A isoforms, both families of scFv recognize antigenic determinan t(s), which was mapped to residues 413-450 of eEF1A-1/2 by Western blot ana lysis of recombinant human eEF1A (hEF1A) fragments. Prior to the Western bl otting analysis, the epitope location had been suggested using a novel appr oach where phage-antibody repertoire derived scFv were used to select phage -displayed peptides. Hereby, peptides containing a SFXD motif, matching the SFSD(414-418) sequence found in hEF1A-1 were isolated. The structure of eu karyotic EF1A from yeast indicates a discontinuous nature of the epitope wi th distal functional elements juxtaposed by the protein fold. Finally, the scFv A8 was applied for immunofluorescence studies of transformed human amn ion cells and MCF-7 fibroblasts. In both cases a perinuclear localization o f hEF1A was observed. No evidence for the reported nuclear localization of hEF1A was obtained.