Antibody-producing Monoclonal B Cell Lines from Multiple Sclerosis Patients Obtained by B Cell Immortalization

Abstract

B cells and oligoclonal antibodies are present in the cerebrospinal fluid (CSF) of MS patients but their target antigens remain unknown. The focus of this study was to produce and characterize antibodies in MS based on B cell immortalization. Antibodies were produced from CSF and peripheral blood mononuclear cells (PBMC) of 7 MS patients and 6 control patients with non-inflammatory neurological disease (NIND/OIND). PBMC or CSF cells were cultured in the presence of irradiated allogeneic PBMC, T cell inhibitory and B cell stimulating factors to activate B cells. Epstein-Barr virus (EBV) was then added to transform B cells which was verified by screening the culture supernatant for the presence of immunoglobolin G. Positive cultures were further cloned and clonality was verified. We obtained 37 immortalized B cell lines from 7 MS patients, 7 originating from CSF of 1 MS patient and 30 from PBMC of 6 MS patients. From 5 NIND and 1 OIND patients 9 B cell lines have been isolated, tow derived from CSF cells of 1 patient and 7 from PBMC of 5 other patients. B cell spectratyping analysis showed that most of the immortalized B cell lines were monoclonal. Preliminary screening demonstrated intracellular binding of antibodies obtained from 8 immortalized B cell lines to a human oligodendroglioma(HOG)cell line. B cell immortalization has proved to be a useful method for the production of antibodies. The obtained monoclonal antibodies will be further analyzed for autoreactivity by detecting antibody binding to healthy and EAE brain tissue from rat and rhesus monkey and to some viruses such as cytomegalovirus(CMV) and EBV.