Oxidized phospholipid ELISA

POVPC/PGPC ELISA protocol was designed and optimized based on previously described protocols^39,40. Briefly, POVPC, PGPC (Cayman Chemical) or the mixture of both in 96% ethanol were added to 96 well microtiter plates (Nunc), 10 μg per well. Ethanol was quickly evaporated under N₂ flow. Ethanol was used as a negative control. The wells were incubated with 150 μl of blocking solution, 10 % heat deactivated fetal bovine serum (hdFBS) in 1 % PEG 8,000 (Sigma) in phosphate buffered saline (PBS) at 37°C for 1 hour followed by three washes in 1 % PEG in PBS.

POVPC-BSA was prepared by mixing fatty acid-free BSA, 1 mg/mL, with POVPC in a glass tube to get a final concentration of POVPC as 10 μg/mL. LDL,/OxLDL/PH-BSA ELISA protocol was performed as previously described²⁷. Ninety-six-well microtiter plate (Nunc) was plated with 100 μL of LDL (Alfa Aesar, BT903), OxLDL (Alfa Aesar, BT-910), PH-BSA (Biosearch Tech, PC-1011-10), or POVPC-BSA (10 μg/mL each) overnight at 4° C. After three washes in PBS, the plate was blocked with 1% BSA (fatty acid-free) at 37°C for 1 hour followed by another three washes in PBS.

Test sera or IgM antibody were diluted as indicated with 10 % hdFBS in 1 % PEG (POVPC/PGPC ELISA) or 1% BSA and aliquoted 50 μl/well onto ELISA plates and incubated at 37°C for 1 hour.After three washes in 1 % PEG in PBS (POVPC/PGPC ELISA) or PBS, peroxidase-coupled anti-mouse (IgG/IgM) secondary antibody (Amersham, NXA931), diluted at 1:2,000 with 10 % hdFBS in 1 % PEG (POVPC/PGPC ELISA) or 1% BSA in PBS, was added to wells and incubated at 37°C for 1 hour. After three washes in 1 % PEG in PBS (POVPC/PGPC ELISA) or in PBS, peroxidase was developed by ABTS™ (Sigma) substrate solution at room temperature for 30 minutes. Absorbance was measured at 405 nm using a SpectraMax 190 (Molecular Devices).