Cultivation and Immortalization of Human B-Cells Producing a Human Monoclonal IgM Antibody Binding to MDA-LDL: Further Evidence for Formation of Atherogenic MDA-LDL Adducts in Humans In Vivo

Oxidatively modified low-density lipoprotein (oLDL) is firmly believed to play an important role in the initiation and development of atherosclerosis, and malonic dialdehyde (MDA) is one of the major lipid peroxidation breakdown products involved in this process. In recent decades, antibodies against MDA-LDL have been detected in human and animal sera. In our study, human B-cells from the peripheral blood of a healthy female donor were fused with the SP2/0 mouse myeloma cell line. Antibody-producing hybridomas were detected by MDA-LDL-IgG/IgM enzyme-linked immunosorbent assays (ELISA) and Cu(++)-oxidized LDL IgG/IgM (oLAb) ELISA. Cells with supernatants emitting positive signals for antibodies were then cloned and after sufficient multiplication frozen and stored under liquid nitrogen. Due to the loss of antibody-producing ability, we established an MDA-LDL-IgM-producing cell line by recloning. This allowed isolation and immortalization of several human B-cells. The human donor had not been immunized with MDA-modified proteins, thus obviously producing MDA-LDL antibodies in vivo. Furthermore, using these antibodies for in vitro experiments, we were able to demonstrate that MDA epitopes are among the epitopes generated during Cu(++)-LDL oxidation as well. Finally, these antibodies compete in ELISA and cell culture experiments with MDA as a challenging toxin or ligand.