EPITOPE MAPPING AND IMMUNONEUTRALIZATION OF RECOMBINANT HUMAN STEM-CELL FACTOR

The epitope regions of three anti-[stem-cell factor (SCF)] Ig have bee n mapped by characterization of immunoreactivities against truncated f orms of SCF in immunoblots and against synthetic peptides in solution- phase competition ELISA, Two of the antibodies, mAb 7H6 and mAb 8H7A, were raised against Escherichia coli-derived human SCF-(1-164) while t he third, polyclonal antibody (pAb) 1337, was raised against a peptide corresponding to residues 3-31 of human SCF. The epitopes of mAbs 7H6 and 8H7A have been mapped to residues 61-95 and 95-110, respectively. The epitope of pAb 1337 has been mapped to residues 21-31, The abilit y of the anti-SCF Ig to recognize E. coli-derived human SCF presented in various formals, i.e. partially denatured (fixed in standard ELISA or on a western blot) or native (in solution), was studied, mAb 7H6 re cognized its epitope in partially denatured or native SCF with equally high affinity, while mAb 8H7A and pAb 1337 recognized their epitopes only when SCF was at least partially denatured. mAb 7H6 was found to n eutralize in vitro SCF-mediated cell proliferation and SCF binding to its receptor, when present in equimolar concentrations relative to the ligand, suggesting that the epitope region is functionally significan t. Evidence that the mAb 7H6 epitope is represented by discontinuous r egions (residues within sequences 61-65 and 91-95 are critically invol ved) is presented, The observation that the mAb 7H6 epitope is discont inuous has implications for the structure of SCF.