title: Fluorescence correlation spectroscopy as a sensitive and useful tool for revealing potential overlaps between the epitopes of monoclonal antibodies on viral particles.
abstract: Although the enzyme-linked immunosorbent assay (ELISA) is well established for quantitating epitopes on inactivated virions used as vaccines, it is less suited for detecting potential overlaps between the epitopes recognized by different antibodies raised against the virions. We used fluorescent correlation spectroscopy (FCS) to detect the potential overlaps between 3 monoclonal antibodies (mAbs 4B7-1H8-2E10, 1E3-3G4, 4H8-3A12-2D3) selected for their ability to specifically recognize poliovirus type 3. Competition of the Alexa488-labeled mAbs with non-labeled mAbs revealed that mAbs 4B7-1H8-2E10 and 4H8-3A12-2D3 compete strongly for their binding sites on the virions, suggesting an important overlap of their epitopes. This was confirmed by the cryo-electron microscopy (cryo EM) structure of the poliovirus type 3 complexed with the corresponding antigen-binding fragments (Fabs) of the mAbs, which revealed that Fabs 4B7-1H8-2E10 and 4H8-3A12-2D3 epitopes share common amino acids. In contrast, a less efficient competition between mAb 1E3-3G4 and mAb 4H8-3A12-2D3 was observed by FCS, and there was no competition between mAbs 1E3-3G4 and 4B7-1H8-2E10. The Fab 1E3-3G4 epitope was found by cryoEM to be close to but distinct from the epitopes of both Fabs 4H8-3A12-2D3 and 4B7-1H8-2E10. Therefore, the FCS data additionally suggest that mAbs 4H8-3A12-2D3 and 4B7-1H8-2E10 bind in a different orientation to their epitopes, so that only the former sterically clashes with the mAb 1E3-3G4 bound to its epitope. Our results demonstrate that FCS can be a highly sensitive and useful tool for assessing the potential overlap of mAbs on viral particles.
authors.affiliation: a Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie , Illkirch , France.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Richert
authors.affiliation: a Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie , Illkirch , France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Humbert
authors.affiliation: b Laboratoire d'Enzymologie et Biochimie Structurales (LEBS) UPR 3082 du CNRS, Campus CNRS , Gif-sur-Yvette Cedex , France.
authors.forename: Eric
authors.initials: E
authors.lastname: Larquet
authors.affiliation: c Sanofi Pasteur , Marcy l'Étoile , France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Girerd-Chambaz
authors.affiliation: c Sanofi Pasteur , Marcy l'Étoile , France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Manin
authors.affiliation: c Sanofi Pasteur , Marcy l'Étoile , France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Ronzon
authors.affiliation: a Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie , Illkirch , France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Mély
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D020285:Cryoelectron Microscopy; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D007091:Image Processing, Computer-Assisted; D017955:Poliovirus / Q000276:immunology*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes
keywords: Cryo-electron microscopy; Poliovirus; epitopes; fluorescence correlation spectroscopy; monoclonal antibody