Expression and immunoreactivity to EBNA-1 and BRRF2. (A) BRRF2 and EBNA-1 RNA transcripts were detected in the B95 cell line (B95) and the EBV-transformed B cell line (BC) by BRRF2- (61 bp) and EBNA-1–specific (107 bp) RT-PCR. To exclude contamination with residual DNA, a control sample without reverse transcription (no RT) was included in the experiment. (B). Expression of full-length and partial BRRF2 RNA in the B95 cell line verified by RT-PCR. (C) BRRF2 expression in E. coli. The BRRF2 protein was cloned as partial (16 kDa) and full-length (58 kDa) protein with a 30-kDa GST tag resulting in 46-kDa partial and 88-kDa full-length band on SDS-PAGE (SDS) after Coomassie staining (left). Western blot (WB) and immunostaining with CSF of a control donor (Ctr; middle) and of a representative MS patient (right) confirmed the specific binding of CSF IgG to the BRRF2 proteins. (D) Immunoreactivity to recombinant BRRF2 (upper panels) and EBNA-1 (lower panels) was investigated by ELISA in CSF (left, 1:5 dilution) and serum (right, 1:100 dilution) of 130 MS patients compared with 115 NIND and 85 OIND patients. The immunoreactivities for each sample are given as OD values. Mean ODs and P values comparing the extent of immunoreactivity by Student’s t test are displayed above each group. Fisher’s exact test was applied to compare the frequencies of reactive patients: *Significant compared with NIND patients; ^#significant compared with OIND patients. RT, reverse transcription.