Abstract
Macrophages play a key role in atherogenesis in part through excessive uptake of oxidized LDL (OxLDL) via scavenger receptors. Binding of OxLDL to macrophages has traditionally been assessed using radiolabeled OxLDL. To allow more efficient and convenient measurements, we developed a nonradioactive binding assay in which biotinylated OxLDL (Bt-OxLDL) is added to macrophages in 96-well microtiter culture plates under various conditions and the extent of binding is determined using solid phase chemiluminescent immunoassay techniques. As examples, we show that Bt-OxLDL displayed high and saturable binding to macrophages in contrast to Bt-LDL, which showed very low binding. In competition assays, unlabeled OxLDL and the anti-OxLDL monoclonal antibody E06 inhibited Bt-OxLDL binding to macrophages in a dose-dependent manner. Specific binding of Bt-OxLDL to ApoE/SR-A/CD36 triple knockout macrophages was reduced by 80% as compared with binding to macrophages from ApoE knockout mice. Binding of Bt-OxLDL to CD36 transfected COS-7 cells showed enhanced saturable binding compared with mock-transfected cells. This assay avoids the use of radioactivity and uses small amounts of materials. It can be used to study binding of OxLDL to macrophages and factors that influence this binding. The techniques described should be readily adaptable to study of other ligands, receptors, and cell types.
Highlights
Macrophages play a key role in atherogenesis in part through excessive uptake of oxidized LDL (OxLDL) via scavenger receptors
The extent of bound ligand, such as biotinylated Copper-induced oxidation of LDL (CuOxLDL) (Bt-CuOxLDL), was determined by the use of alkaline phosphatase conjugated to avidin, which acts on the added LumiPhos 530 detection substrate to produce chemiluminescence, which was measured by a plate reader
Chief among these was finding the optimal number of cells to be plated per well that would lead to optimal receptor expression at the time of the binding assay, when plates were put on ice to inhibit internalization of bound ligand. This variable was dependent on the number of cells seeded per well initially, the media used, and the length of time in culture before actual performance of the binding assay. These conditions varied for the different macrophages studied and the optimal number of cells initially seeded per well, media used, and time in culture to achieve approximately 100% confluence at time of conduct of binding assay were established for J774 cells, Bone marrow-derived macrophages (BMDM), Thioglycollateelicited peritoneal macrophages (TGEPM), and CD36transfected COS-7 cells
Summary
Macrophages play a key role in atherogenesis in part through excessive uptake of oxidized LDL (OxLDL) via scavenger receptors. Binding of Bt-OxLDL to CD36 transfected COS-7 cells showed enhanced saturable binding compared with mock-transfected cells This assay avoids the use of radioactivity and uses small amounts of materials. Development and application of a nonradioactive binding assay of oxidized low-density lipoprotein to macrophage scavenger receptors. Understanding the mechanisms responsible for binding and uptake of OxLDL by macrophages could lead to novel therapeutic approaches to inhibit foam cell formation and atherogenesis. We have demonstrated that various oxidation specific monoclonal antibodies that bind to relevant epitopes on OxLDL can prevent OxLDL binding to macrophage scavenger receptors in culture conditions and when expressed in excess in vivo in murine models of atherosclerosis can inhibit progression of atherosclerosis [4, 5].
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
