Gold Nanoparticle-Conjugated Anti-Oxidized Low-Density Lipoprotein Antibodies for Targeted Lipidomics of Oxidative Stress Biomarkers

Oxidized low-density lipoproteins (OxLDLs), in particular, oxidized phosphatidylcholines (OxPCs), are known to be involved in pathophysiological processes such as cardiovascular diseases and are described as potential biomarkers, for example, for atherosclerosis. In our study, we used the specific affinity of anti-OxLDL antibodies (Abs) conjugated to gold nanoparticles (GNPs) for extraction and enrichment of OxPCs via selective trapping of OxLDLs from plasma combined with the sensitive detection by liquid chromatography/tandem-mass spectrometry (LC-MS/MS). Successful bioconjugation chemistry of Abs via a bifunctional polyethylene glycol (PEG) spacer and protein G linkage, respectively, was controlled by measuring the surface plasmon resonance (SPR) spectra, size, and zeta potentials. Furthermore, the amount of Ab immobilized onto GNP via the PEG linker was determined. With the optimized immobilization chemistry, the ability and potential of the GNP-based extraction procedure was used for the determination of the dissociation constant, K-d, of the OxLDL binding to the GNP-Ab conjugate. Moreover, apparent K-d's were determined for individual PCs and their oxidation products using the compound-specific selected reaction monitoring mode, which allows the characterization of the Ab affinity and, thus, assessment of the potential antigenicity of (Ox)PCs bound to OxLDLs. In summary, the application of GNP-based bioanalysis for selective targeting of OxLDLs and the fast and sensitive detection by LC-MS/MS offers new possibilities for targeted lipidomics in lipoproteins as well as for oxidative stress lipid biomarker screening.