title: Screening random peptide libraries with subacute sclerosing panencephalitis brain-derived recombinant antibodies identifies multiple epitopes in the C-terminal region of the measles virus nucleocapsid protein.
abstract: Infectious and inflammatory diseases of the CNS are often characterized by a robust B-cell response that manifests as increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands. We previously used laser capture microdissection and single-cell PCR to analyze the IgG variable regions of plasma cells from the brain of a patient with subacute sclerosing panencephalitis (SSPE). Five of eight human IgG1 recombinant antibodies (rAbs) derived from SSPE brain plasma cell clones recognized the measles virus (MV) nucleocapsid protein, confirming that the antibody response in SSPE targets primarily the agent causing disease. In this study, as part of our work on antigen identification, we used four rAbs to probe a random phage-displayed peptide library to determine if epitopes within the MV nucleocapsid protein could be identified with SSPE brain rAbs. All four of the SSPE rAbs enriched phage-displayed peptide sequences that reacted specifically to their panning rAb by enzyme-linked immunosorbent assay. BLASTP searches of the NCBI protein database revealed clear homologies in three peptides and different amino acid stretches within the 65 C-terminal amino acids of the MV nucleocapsid protein. The specificities of SSPE rAbs to these regions of the MV nucleocapsid protein were confirmed by binding to synthetic peptides or to short cDNA expression products. These results indicate the feasibility of using peptide screening for antigen discovery in central nervous system inflammatory diseases of unknown etiology, such as multiple sclerosis, neurosarcoidosis, or Behcet's syndrome.
authors.affiliation: Department of Neurology, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, Mail Stop B182, Denver, CO 80262, USA.
authors.forename: Gregory P
authors.initials: GP
authors.lastname: Owens
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Shearer
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Yu
authors.forename: Alanna M
authors.initials: AM
authors.lastname: Ritchie
authors.forename: Kathryne M
authors.initials: KM
authors.lastname: Keays
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Bennett
authors.forename: Donald H
authors.initials: DH
authors.lastname: Gilden
authors.forename: Mark P
authors.initials: MP
authors.lastname: Burgoon
mesh_terms: D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000235:genetics*; D019295:Computational Biology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics*; D006801:Humans; D008459:Measles virus / Q000235:genetics*; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000235:genetics*; D019151:Peptide Library; D010455:Peptides / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013344:Subacute Sclerosing Panencephalitis / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D000939:Epitopes; D019590:Nucleocapsid Proteins; D019151:Peptide Library; D010455:Peptides