Increased IgG and oligoclonal bands (OGBs) are found in the cerebrospinal fluid (CSF) of humans with chronic infectious CNS diseases such as neurosyphilis, cryptococcal and tuberculous meningitis, Lyme disease, some viral meningitides, varicella zoster virus vasculopathy, and subacute sclerosing panencephalitis (SSPE), a chronic encephalitis caused by measles virus (MV). Studies in which the specificity of CSF OGBs was analyzed showed that the antibodies were directed against the agent that causes disease (1-8). For example, the OGBs in SSPE CSF and brain are antibodies directed against MV (1). These studies have led to the hypothesis that the oligoclonal IgG in the brain and CSF of patients with a chronic inflammatory CNS disease of unknown etiology, such as multiple sclerosis (MS), sarcoidosis, and Behcet's disease, is antibody directed against the agent that causes disease. Although OGBs are found in 88-100% of CSF from MS patients (9), the corresponding antigens remain unknown (10, 11). Such unanswered questions point to the need for improved techniques to identify disease-relevant antibodies and their cognate antigens and suggest the promise of such techniques in revealing the causes of inflammatory diseases with unknown etiologies.Here, we used laser-capture microdissection (LCM) to isolate individual CD38ϩ plasma cells from the brain of a patient with SSPE. Single-cell RT-PCR was used to amplify individual IgG heavy (H) and light (L) chain sequences expressed by each cell. Based on overrepresented Ig sequences, we constructed functional recombinant antibodies and identified their target antigens.Materials and Methods SSPE Brain. Brain removed from a 14-year-old male SSPE patient 5 h after death was flash-frozen and stored at Ϫ70°C. Sections (7 m) of frozen SSPE brain were prepared on nonplus glass slides (Fisher Scientific) at Ϫ30°C, fixed in acetone for 5 min at Ϫ20°C, and immunostained at 0°C for CD38ϩ cells. Briefly, sections were treated with 0.1% H 2 O 2 for 30 sec, incubated for 2 min with PBS containing 10% goat serum and then with a 1:50 dilution of mouse anti-human CD38 antibody (DakoCytomation, DAKO) for 10 min, rinsed in PBS, and incubated for 5 min with a 1:100 dilution of horseradish peroxidase-conjugated horse anti-mouse antibody (Vector Laboratories) and 5% goat serum in PBS. After rinsing in PBS, sections were incubated for 5 min with DAB substrate (Vector Laboratories), counterstained with hematoxylin, dehydrated in nuclease-free graded alcohols to 100% ethanol, and cleared with two rinses of H 2 0-free xylene. To preserve RNA, all aqueous solutions contained 200 units͞ml RNase inhibitor (Fisher Scientific), and the total time of tissue exposure to aqueous solutions was Ͻ25 min at 0°C. Staining was visualized by light microscopy.LCM and RT-PCR. LCM was performed on a PixCell IIe microscope (Arcturus Engineering, Mountain View, CA) with CapSure HS caps by using a pulse power of 70 mW, a 7.5-m laser spot diameter, pulse duration of 5 ms, and target voltage of 170 mV. Individual CD38ϩ cells ...