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 ANTIPEPTIDE ANTIBODIES

We produced polyclonal and monoclonal antibodies (MAbs) against recomb inant human (rh) granulocyte-macrophage colony-stimulating factor (GM- CSF) and performed studies of epitope mapping by ELISA, using five syn thetic peptides corresponding to sequences along this molecule. Additi onally, anti-peptide MAbs were generated. The antibody ability to inhi bit rhGM-CSF activity was determined using as bioassay the MO7e cell l ine, which is dependent on hGM-CSF for growth in vitro. An immunodomin ant 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 alpha-helix o f the molecule, was recognized by neutralizing anti-protein antibodies . Similarly, MAbs anti- 14-24 cross-reacted with rhGM-CSF and specific ally blocked its function. Replacement of Val(16) or Asn(17) with alan ine greatly reduced the antibody-binding capacity to peptide 14-24, wh ereas substitution of Gln(20) or Glu(21) was less critical. Monoclonal antibodies generated against residues 30-41 (corresponding to an intr ahelical loop) and 79-91 (homologous to a sequence including part of t he third alpha-helix) or its analog [Ala(88)](79-91)beta Ala-Cys, were conformation dependent and nonneutralizing: they failed to react or b ound poorly to rhGM-CSF in ELISA, but readily recognized the homologou s sequence in the denatured protein, by Western blotting,