title: Gold nanoparticle-antibody conjugates for specific extraction and subsequent analysis by liquid chromatography-tandem mass spectrometry of malondialdehyde-modified low density lipoprotein as biomarker for cardiovascular risk.
abstract: Oxidized low-density lipoproteins (OxLDLs) like malondialdehyde-modified low-density lipoprotein (MDA-LDL) play a major role in atherosclerosis and have been proposed as useful biomarkers for oxidative stress. In this study, gold-nanoparticles (GNPs) were functionalized via distinct chemistries with anti-MDA-LDL antibodies (Abs) for selective recognition and capture of MDA-LDL from biological matrices. The study focused on optimization of binding affinities and saturation capacities of the antiMDA-LDL-Ab-GNP bioconjugate by exploring distinct random and oriented immobilization approaches, such as (i) direct adsorptive attachment of Abs on the GNP surface, (ii) covalent bonding by amide coupling of Abs to carboxy-terminated-pegylated GNPs, (iii) oriented immobilization via oxidized carbohydrate moiety of the Ab on hydrazide-derivatized GNPs and (iv) cysteine-tagged protein A (cProtA)-bonded GNPs. Depending on immobilization chemistry, up to 3 antibodies per GNP could be immobilized as determined by ELISA. The highest binding capacity was achieved with the GNP-cProtA-Ab bioconjugate which yielded a saturation capacity of 2.24±0.04μgmL(-1) GNP suspension for MDA-LDL with an affinity Kd of 5.25±0.11×10(-10)M. The GNP-cProtA-antiMDA-LDL bioconjugate revealed high specificity for MDA-LDL over copper(II)-oxidized LDL as well as native human LDL. This clearly demonstrates the usefulness of the new GNP-Ab bioconjugates for specific extraction of MDA-LDL from plasma samples as biomarkers of oxidative stress. Their combination as specific immunoextraction nanomaterials with analysis by LC-MS/MS allows sensitive and selective detection of MDA-LDL in complex samples.
authors.affiliation: Department of Analytical Chemistry, University of Vienna, Währingerstrasse 38, 1090 Vienna, Austria.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Haller
authors.affiliation: Department of Analytical Chemistry, University of Vienna, Währingerstrasse 38, 1090 Vienna, Austria.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Lindner
authors.affiliation: Institute of Pharmaceutical Sciences, Pharmaceutical (Bio)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany. Electronic address: Michael.laemmerhofer@uni-tuebingen.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Lämmerhofer
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry / Q000276:immunology; D015415:Biomarkers / Q000097:blood*; D002318:Cardiovascular Diseases / Q000097:blood / Q000175:diagnosis; D002853:Chromatography, Liquid / Q000379:methods*; D003429:Cross Reactions; D003545:Cysteine / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D008077:Lipoproteins, LDL / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D008315:Malondialdehyde / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry*; D012307:Risk Factors; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D055910:Antibodies, Immobilized; D015415:Biomarkers; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D008315:Malondialdehyde; D003545:Cysteine
keywords: Antibody bioconjugate; Gold nanoparticle; High-performance liquid chromatography–tandem mass spectrometry; Malondialdehyde-modified low-density lipoprotein; Oxidative stress; Oxidized low-density lipoprotein