Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform.
Sci Rep
; 6: 31458, 2016 08 17.
Article
en En
| MEDLINE
| ID: mdl-27530334
ABSTRACT
We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete "hot spots" with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Bacteriófago lambda
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Biblioteca de Péptidos
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Anticuerpos Monoclonales de Origen Murino
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Secuenciación de Nucleótidos de Alto Rendimiento
/
Epítopos
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Sci Rep
Año:
2016
Tipo del documento:
Article
País de afiliación:
Italia