Immortalization was verified 28 days after seeding by screening the culture supernatant for antibody production using dot blot analysis and by light microscopic examination of cell growth. disease markers or targets for therapy. Keywords:B cell immortalization, Epstein-Barr virus, Monoclonal antibodies, B cell spectratyping == 1. Introduction == B cell immortalization by Epstein-Barr virus (EBV) is an established method for antibody production. EBV infects B cells via their CD21 receptor[1]and subsequently transforms them into continually dividing, lymphoblastoid cell lines that produce antibodies representing the humoral immune response in vivo. Antibodies can be generated against an infectious agent or tumour cells, rendering the resulting antibodies attractive for therapy. In addition, B cell immortalization can be a valuable tool for the production and characterization of autoreactive antibodies from patients with autoimmune diseases. This can provide more insight into the underlying mechanisms of humoral immune responses in autoimmunity and can lead to the identification of new autoantigens, disease markers or targets for therapy[2],[3],[4]. The major advantage of B cell immortalization, when compared to other antibody-producing techniques[5],[6],[7],[8],[9],[10], is the generation of fully human antibodies that truly reflect both the specificity and diversity of the human immune response, generated from the human B cell repertoire, without the need for specific immunization. The original B cell immortalization technology, first described in 1977, was performed by culturing B cells in the presence of EBV, obtained from the marmoset lymphocyte cell line B95-8[11],[12]. The resulting immortalized B cell lines were mainly unstable immunoglobulin M (IgM) producing clones with low affinity. Various adjustments to the procedure have been tried since then, but immortalization and B cell growth rate remained inefficient. More recently, antibodies neutralizing SARS coronavirus and cytomegalovirus (CMV) were produced successfully by the introduction of the polyclonal B cell activator CpG2006 in the B cell immortalization process or by B cell activation prior to EBV infection, respectively[13],[14]. In our hands, these Vilazodone D8 methods resulted in a low reproducibility. The current study was aimed at developing a B cell immortalization procedure with a high efficiency and reproducibility when seeding low B cell numbers per well, that could easily be adopted for the production of (autoreactive) antibodies from patients with autoimmune disease. Such a method can be especially advantageous when autoreactive B cells are not easily available, for example in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients or the synovial fluid of rheumatoid arthritis (RA) patients. Moreover, seeding low Vilazodone D8 B cell numbers per well limits the bias towards the preferential outgrowth of fast growing immortalized B cells. == 2. Materials and methods == == 2.1. B cell immortalization procedure == Peripheral blood from healthy donors was obtained with informed consent. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Hypaque density gradient centrifugation (SigmaAldrich). To isolate IgG+CD22+B cells, PBMC were stained with Vilazodone D8 PE-Cy5-labelled anti-CD22 antibodies (Ab) and PE-labelled anti-IgG Ab for 30 min at 4 C and subsequently enriched by means of FACS sorting using a FACSAria II cell sorter (all from BD Biosciences). The FACS sorted cells were immediately used for B cell immortalization assays. IgG+CD22+B cells were cultured in U-bottom 96-well plates (Nunc) at 50 cells per well in RPMI 1640 medium supplemented withl-glutamine, 10 mM HEPES buffer, 1 mM sodium Rabbit Polyclonal to TAS2R12 pyruvate, 50 U/ml penicillin, 50 g/ml streptomycin (all from Invitrogen Life Technologies) and 10% heat-inactivated fetal bovine serum (FBS, HyClone). All immortalization experiments were started in 30 wells for each tested condition. The isolated B cells were immortalized during 2 weeks in the presence of 1 105autologous irradiated (83 Gy) feeder cells, 30% v/v EBV-containing supernatant (3.4 108viral copies/ml) of the B95-8 cell line (ATCC) and 1 g/ml CpG2006 (ODN2006, 5-tcgtcgttttgtcgttttgtcgtt-3, InvivoGen) (Fig. 1). Next, cells were restimulated during 7 days with 1 g/ml CpG2006 together with 50 U/ml IL-2 (Roche Diagnostics). Culture medium was then replaced and cells were continuously cultured without extra stimuli. Immortalization was verified 28 days after seeding by screening the culture supernatant for antibody production using dot blot analysis and by light microscopic examination of cell growth. Both parameters were included since positive antibody measurements were sometimes observed in the absence of B cell growth, due to B cell activation at the start of the culture. == Fig. Vilazodone D8 1. == B cell immortalization.