Measurement of MDA-LDL and ApoB by Sandwich ELISA

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).