We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb) epitopes. For this purpose, we used a novel mAb (designated 31E10/E7) directed against Neisserial Heparin-Binding Antigen (NHBA), a component of the anti-group B meningococcus Bexsero1 vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128) in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.
Titolo: | Epitope mapping of a monoclonal antibody directed against neisserial heparin binding antigen using next generation sequencing of antigen-specific libraries |
Autori: | DOMINA, MARIA (Primo) BENINATI, Concetta (Ultimo) |
Data di pubblicazione: | 2016 |
Rivista: | |
Abstract: | We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb) epitopes. For this purpose, we used a novel mAb (designated 31E10/E7) directed against Neisserial Heparin-Binding Antigen (NHBA), a component of the anti-group B meningococcus Bexsero1 vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128) in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods. |
Handle: | http://hdl.handle.net/11570/3094558 |
Appare nelle tipologie: | 14.a.1 Articolo su rivista |
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