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.

Franz Tatzber,Ulrike Resch,Meinrad Lindschinger,Sandra Holasek,Roswitha Pfragner,Edith Pursch,Willibald Wonisch,Gerhard Cvirn

doi:10.1155/2017/6047142

Abstract

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.

Highlights

Acute myocardial infarction (AMI) and various malignancies are the most frequent causes of death in Europe and the United States of America (USA), and lipid peroxidation (LPO) due to oxidative stress appears to be importantly involved in various stages of these disease processes
This leads to formation of reactive oxygen species (ROS) and various highly reactive aldehydes [3, 4], which in turn build up Oxidative Medicine and Cellular Longevity adducts with amino acids of proteins, for example in the case of low-density lipoprotein (LDL) apolipoprotein B (Apo B)
After the clones were first tested by enzyme-linked immunosorbent assays (ELISA) on day 7, 116 out of 480 wells were identified as giving positive signals for human IgG antibodies binding to Oxidatively modified low-density lipoprotein (oLDL)

Summary

Introduction

Acute myocardial infarction (AMI) and various malignancies are the most frequent causes of death in Europe and the United States of America (USA), and lipid peroxidation (LPO) due to oxidative stress appears to be importantly involved in various stages of these disease processes. LPO is at least one basic process leading to oLDL by consumption of protective antioxidants present on each low-density lipoprotein (LDL) particle and subsequent initiation of free radical-mediated chain reactions. This leads to formation of reactive oxygen species (ROS) and various highly reactive aldehydes [3, 4], which in turn build up Oxidative Medicine and Cellular Longevity adducts with amino acids of proteins, for example in the case of LDL apolipoprotein B (Apo B). In 2001, the first human monoclonal antibody was described which recognizes epitopes of oxidized LDL [17, 18] This group isolated RNA from peripheral mononuclear cells and prepared cDNA to construct a phage display antibody library. Using hMCA for diagnostic purposes in vitro or in cell cultures would make models possible consisting strictly of human material, so reducing the possibility of unwanted unspecific effects

Methods

Results

Conclusion