Cell Line EL-4-B5 is a subclone of murine EL4 thymoma that is a bromo-deoxyufidine-resistant mutant, and can be used as a feeder layer to generate B cell cultures yielding human monoclonal antibodies.
IL-2 producing EL-4 mouse thymoma cells underwent mutagenesis with ethyl-methane-sulfonate resulting in fusion partners for T cell hybridomas. It was then discovered that the thymidine kinase-deficient and ouabaine-resistant clone EL-4 BurOUr 6.1 also activated murine and human B cells via cell contact. Subcloning produced the EL-4-B5 cells, which strongly activate B cells. EL4-B5 is grown with B cells to activate the B cells via direct cell contact to induce proliferation, differentiation, and secretion of antibody. The EL-4-B5 cell line offers a method for the generation of recombinant human mAbs from single antigen-specific B cell clones selected with fluorescent VLPs and can be used to generate human mAbs to many other viruses whose proteins can self-assemble into VLPs.
From the laboratory of James E. Crowe, Jr., MD, Vanderbilt University.
|Product Type:||Cell Line|
|Cell Type:||Mouse thymoma continuous cell line|
|Growth Conditions:||RPMI-1640, 10% FBS heat inactivated, with 2 mM glutamine, usually antibiotics (typically pen/strep or gentamicin plus amphotericin B), 10 mM HEPES, 2- mercaptoethanol (0.056 mM)|
|Subculturing:||Cells are both adherent and suspension. The suspension cells are the more healthy ones. Can tolerate large splits after line is established (volume ratios of 1:10, for example). Helper function is optimal when cells are in log phase growth. Keep density below 106cells per mL at all times|
|Cryopreservation:||Standard conditions (typically 10% DMSO, 90% FBS)|
Human lymphocytes identified by flow cytometry to be double-positive for CD19-PE and RV VLP-GFP were single cell sorted into 96-well plates (one cell per well) and expanded in culture using a feeder-cell layer and cytokine combinations as indicated. Production of total Ig and RV VP6- or VP7-specific Ig was detected by ELISA using supernatants of the B cell cultures. Antibody VH and VL regions were rescued by nested RT-PCR followed by TA cloning. Nucleotide sequences were analyzed, unique VH and VL regions were subcloned into a novel Fab expression vector, and subsequently expressed as Fabs.
Adapted from: Weitkamp J-H., et al. J Immunol Methods 2003, 275:223-237.
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