Biotinylated anti apob antibody

All samples were anonymised and personnel conducting assays were fully blinded to the patient treatment allocation. ELISAs to detect MDA-LDL and anti-oxLDL antibodies were performed as previously described [13 (link)]. Levels of MDA-LDL were measured using 10 μg/mL LO1, a novel laboratory-developed monoclonal IgG3κ murine antibody, as the capture antibody in a sandwich enzyme-linked immunosorbent assay (ELISA). LO1 reacts with MDA-LDL but minimally with native LDL [14 (link)]. A biotinylated anti-ApoB antibody (Abcam, Cambridge, MA, USA) at 1:2000 dilution and horseradish peroxidase (HRP)-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA) at 1:200 dilution were used for detection. Subsequently, for this and all other ELISAs, 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma Aldrich, Poole, UK) was added and the reaction stopped with 0.5 M H2SO4. Plates were read at an optical density (OD) of 450 nm using a Synergy HT microplate reader (BioTek, Winooski, VT, USA).

Sandwich ELISA protocols were established to measure the majority of components within our study. MDA-LDL was measured using LO1 (10 μg/mL) as the capture antibody for MDA-LDL [18 (link)], whilst polyclonal goat anti-human anti-ApoB (Abcam, Cambridge, MA, USA; 1:2000 dilution) was used as the capture antibody for ApoB. A biotinylated anti-ApoB antibody (Abcam, Cambridge, MA, USA; 1:2000), followed by horseradish peroxidase (HRP)-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA; 1:200) were used for detection of both MDA-LDL and ApoB. For these assays, and all other ELISAs, development was achieved by adding 3,3′,5,5′-tetramethylbenzidine (Sigma Aldrich, Poole, UK), after which the reaction was stopped with 0.5 M H₂SO₄. Plates were read at an optical density of 450 nm using a Synergy HT microplate reader (BioTek, VT, USA).