Identification of the CSF IgG-binding epitope. (A) Peptide scan analysis with 13-mer peptides that overlapped 11 AAs, covering the entire sequence of protein B3 of pattern I (upper membrane) and protein H5 of pattern II (lower membrane), was used to define the epitopes. Membranes were incubated with CSF (in 1:100 dilution) from MS patients immunoreactive to B3 or H5. Binding of IgG was visualized by anti-human IgG-HRP and TMB substrate. The minimal peptide epitopes were EPARSRSR for motif 1 and EAGAGGGA for motif 2. Similar results were obtained with CSF from 2 additional patients. (B) Substitution analysis was performed in order to define the optimal binding motif for the 8 AA epitopes defined in A. Binding of IgG was visualized by anti-human IgG-HRP and TMB substrate. A representative example for pattern I is shown. 1–8, the substituted AA-positions of the minimal epitope; 1–20, the 20 naturally occurring acids A–Y; *original peptide sequence. Similar results were obtained with 2 additional CSF samples from MS patients. (C) Definitions of 2 consensus motifs were based on the epitope mapping in 3 MS patients. These motifs were used to search the Swiss-Prot database. Database searching revealed 10 proteins matching with motif 1 and 13 proteins with motif 2. Two identified EBV proteins and the genomic locations according to http://www.ncbi.nlm.nih.gov are displayed. (D) Qualitative comparison of CSF IgG binding to peptides matching motif 1 (left) and motif 2 (right). Antibody binding was quantified by gel densitometry (highest signal and integrated density), which revealed the strongest binding to the 2 EBV epitopes BRRF2 and EBNA-1. The analysis was performed with similar results in 2 additional patients.