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.
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 MO7e 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 alpha-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 alpha-helix) or its analog [Ala88](79-91)beta Ala-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.
Beffy
P
P
Institute of Mutagenesis and Differentiation, C.N.R., Pisa, Italy.
Rovero
P
P
Di Bartolo
V
V
Laricchia Robbio
L
L
Dané
A
A
Pegoraro
S
S
Bertolero
F
F
Revoltella
R P
RP
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D016056:Immunodominant Epitopes / Q000276:immunology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor