title: Development and application of a nonradioactive binding assay of oxidized low-density lipoprotein to macrophage scavenger receptors.
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.
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA, 92093-0682.
authors.forename: Erica N
authors.initials: EN
authors.lastname: Montano
authors.forename: Agnès
authors.initials: A
authors.lastname: Boullier
authors.forename: Felicidad
authors.initials: F
authors.lastname: Almazan
authors.forename: Christoph J
authors.initials: CJ
authors.lastname: Binder
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Witztum
authors.forename: Karsten
authors.initials: K
authors.lastname: Hartvigsen
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008163:Luminescent Measurements; D051379:Mice; D011485:Protein Binding; D051116:Receptors, Scavenger / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D008077:Lipoproteins, LDL; D051116:Receptors, Scavenger; C099252:oxidized low density lipoprotein
keywords: CD36; SR-A; atherosclerosis; macrophages; monoclonal antibody E06; oxidized LDL