Epitope mapping and immunoneutralization of recombinant human stem-cell factor.
The epitope regions of three anti-[stem-cell factor (SCF)]g have been mapped by characterization of immunoreactivities against truncated forms 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 the 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 ability of the anti-SCF Ig to recognize E. coli-derived human SCF presented in various formats, i.e. partially denatured (fixed in standard ELISA or on a western blot) or native (in solution), was studied, mAb 7H6 recognized 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 neutralize 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 significant. Evidence that the mAb 7H6 epitope is represented by discontinuous regions (residues within sequences 61-65 and 91-95 are critically involved) is presented. The observation that the mAb 7H6 epitope is discontinuous has implications for the structure of SCF.
Mendiaz
E A
EA
Amgen Inc., Amgen Center, Thousand Oaks, CA 91320-1789, USA.
Chang
D G
DG
Boone
T C
TC
Grant
J R
JR
Wypych
J
J
Aguero
B
B
Egrie
J C
JC
Langley
K E
KE
D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D008533:Megakaryocytes / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D010455:Peptides / Q000276:immunology; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology; D017384:Sequence Deletion; D019089:Stem Cell Factor / Q000031:analogs & derivatives / Q000276:immunology*
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit