THE USE OF LIGHT-DIRECTED COMBINATORIAL PEPTIDE-SYNTHESIS IN EPITOPE MAPPING

The application of light-directed combinatorial peptide synthesis to e pitope mapping is described Photolithography and solid phase peptide s ynthesis were combined in an automated fashion to assemble arrays cont aining 1024 peptide sequences on a glass support in ten steps with the precise location of each peptide known. The simultaneous synthesis of two slides containing three arrays of peptides each allowed for the i ndependent screening of both a monoclonal( antibody (mAb) and its Fab fragment at two different concentrations A binary synthesis strategy w as used to assemble the arrays, resulting in all deletions and truncat ions possible within the FLRRQFKVVT sequence being present and availab le for screening. The relative binding interactions of each peptide wa s determined by incubating the arrays with either mAb D32.39 and gent antimouse immunoglobulin G-FITC or mAb D32.39 Fab-FITC conjugate, foll ow ed by scanning the surface for fluorescence with an epifluorescence microscope. The fragment RQFKVVTT was found to bind tightly to both t he mAb and Fab fragment while tethered to the surface, and was measure d to have 0.49 nM affinity in solution. The frame-shifted RRQFKVV sequ ence was found to have lower affinity both in solution (1.3 mM) and on the surface. The fragment RQFKVV was determined to be responsible for antibody recognition and was found to bind tightly when tethered to t he surface, yet exhibited no binding in solution as the free acid, sug gesting the requirement of an amidated C-terminus or an additional fla nking residue. A deletion analysis revealed that the novel RQFKVT sequ ence exhibited higher affinity than the RQFKVV sequence while tethered to the surface. (C) 1995 John Wiley & Sons, Inc.