An Immunodominant Epitope in a Functional Domain Near the N-Terminus of Human Granulocyte-Macrophage Colony-Stimulating Factor Identified by Cross-Reaction of Synthetic Peptides with Neutralizing Anti-Protein and Anti-Peptide Antibodies

Abstract

ABSTRACT

We produced polyclonal and monoclonal antibodies (MAbs) against recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and performed studies of epitope mapping by ELISA, using five synthetic peptides corresponding to sequences along this molecule. Additionally, anti-peptide MAbs were generated. The antibody ability to inhibit rhGM-CSF activity was determined using as bioassay the M07e cell line, which is dependent on hGM-CSF for growth in vitro. An immunodominant epitope able to induce the highest neutralization antibody titers was identified near the N terminus of hGM-CSF. A synthetic peptide 14–24, homologous to a sequence including part of the first α-helix of the molecule, was recognized by neutralizing anti-protein antibodies. Similarly, MAbs anti- 14–24 cross-reacted with rhGM-CSF and specifically blocked its function. Replacement of Val¹⁶ or Asn¹⁷ with alanine greatly reduced the antibody-binding capacity to peptide 14–24, whereas substitution of Gln²⁰ or Glu²¹ was less critical. Monoclonal antibodies generated against residues 30–41 (corresponding to an intrahelical loop) and 79–91 (homologous to a sequence including part of the third α-helix) or its analog [Ala⁸⁸](79–91)βAla-Cys, were conformation dependent and nonneutralizing: they failed to react or bound poorly to rhGM-CSF in ELISA, but readily recognized the homologous sequence in the denatured protein, by Western blotting.