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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

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

P Beffy, P Rovero, V Di Bartolo, L Laricchia Robbio, A Dané, S Pegoraro, F Bertolero, R P Revoltella

Hybridoma. 1994 Dec;13(6):457-68.

PMID: 7737670

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 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4246483	Anti-Protein C antibody produced in rabbit			Price

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