Specific intrathecal IgG response to EBV proteins. (A) Intrathecal IgG response to BRRF2 proteins in MS patients and control donors detected by ELISA. CSF and serum were adjusted to 10 mg/l IgG and the ratio of OD CSF/OD serum determined. Ratios above 1.2 indicate intrathecal synthesis. MS patients showed intrathecal BRRF2-specific IgG synthesis more frequently than controls. (B) IEF-immunoblot demonstrating specific binding of CSF oligoclonal IgG bands from 2 MS patients to the EBV proteins. The membranes were coated with BRRF2, EBNA-1, or a solution containing 10% milk alone as indicated. Affinity-blotted IgG was detected with anti-human IgG-HRP and visualized by TMB substrate. (C) IEF-immunoblot for BRRF2-specific OCBs (right) and total OCB pattern (left). Detection of bound IgG was performed as described in B. BRRF2-specific OCBs correspond to some of the major bands in the OCB pattern of the MS patient. (D) Loss of OCBs by preabsorption with EBNA-1 but not GAPDH. 2D-electrophoresis and IgG immunoblot of CSF from a patient with EBNA-1 immunoreactivity. No, no preabsorption of CSF-IgG was performed with EBNA-1 or GAPDH. (E) Solution phase assays demonstrate high affinity and specificity of CSF antibodies to EBNA-1 (left) and BRRF2 (right). Soluble EBV proteins at different dilutions were incubated with CSF and, subsequently, the remaining immunoreactivity measured by ELISA. Competition by soluble antigens is displayed. No competition was measured with GAPDH protein. For each assay, 4 MS patients were analyzed. *Fisher's exact probability test.