The use of light-directed combinatorial peptide synthesis in epitope mapping.
The application of light-directed combinatorial peptide synthesis to epitope mapping is described. Photolithography and solid phase peptide synthesis were combined in an automated fashion to assemble arrays containing 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 independent screening of both a monoclonal antibody (mAb) and its Fab fragment at two different concentrations. A binary synthesis strategy was used to assemble the arrays, resulting in all deletions and truncations possible within the FLRRQFKVVT sequence being present and available for screening. The relative binding interactions of each peptide was determined by incubating the arrays with either mAb D32.39 and goat antimouse immunoglobulin G-FITC or mAb D32.39 Fab-FITC conjugate, followed by scanning the surface for fluorescence with an epifluorescence microscope. The fragment RQFKVVT was found to bind tightly to both the mAb and Fab fragment while tethered to the surface, and was measured to have 0.49 nM affinity in solution. The frame-shifted RRQFKVV sequence 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 the surface, yet exhibited no binding in solution as the free acid, suggesting the requirement of an amidated C-terminus or an additional flanking residue. A deletion analysis revealed that the novel RQFKVT sequence exhibited higher affinity than the RQFKVV sequence while tethered to the surface.
Holmes
C P
CP
Affymax Research Institute, Palo Alto, CA 94304.
Adams
C L
CL
Kochersperger
L M
LM
Mortensen
R B
RB
Aldwin
L A
LA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics; D005453:Fluorescence; D016368:Frameshift Mutation; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000138:chemical synthesis*; D017421:Sequence Analysis
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D010455:Peptides