Microfluor plates

Plasma antibody levels from human and mouse were measured with a chemiluminescence immunoassay (enzyme-linked immunosorbent assay, ELISA). Antigen was dissolved in PBS (5 μg/ml) and incubated at +4°C overnight in MicroFluor plates (Thermo Scientific, Rockford, IL, USA). Automated plate washer was used to wash the plates three times with PBS containing 0.27mM ethylenediaminetetraacetic acid (EDTA). Plates were blocked with PBS-EDTA containing 0.5% fish gelatin (Fg) and incubated for one hour at room temperature. Plasma samples were diluted with 0.5% Fg-PBS-EDTA and the antibody levels were measured with alkaline phosphatase-labeled antibodies (anti-human-IgG, anti-human-IgM, anti-mouse-IgG, anti-mouse-IgM) diluted according to manufacturer specifications (Sigma-Aldrich). Chemiluminescence was detected using LumiPhos 530 (33% Lumigen) substrate, measured with luminescence multilabel counter (PerkinElmer Victor³V), and expressed in relative light units (RLU).
Specific binding of plasma antibodies to Aa-HSP60 and MAA-LDL was tested with competitive chemiluminescence immunoassay. Aa-HSP60 and MAA-LDL (0–100 μg/mL) were incubated with plasma samples overnight at +4°C. Incubated solutions were centrifuged at 16,000 × g at +4°C and the remaining antibody levels measured with ELISA as described above.

The amount of antibodies was determined by chemiluminescence immunoassay as described elsewhere [21 (link)]. Rgp44, heat-inactivated Pg, MAA-LDL, CuOx-LDL, phosphocholine-conjugated bovine serum albumin (PCho-BSA, Biosearch Technologies, Novato, CA, USA), BSA, Pg-LPS (Invivogen, San Diego, CA, USA) were used as antigens. The antigens were coated (5 μg/mL) in phosphate-buffered saline (PBS) with 0.27 mmol/L ethylenediaminetetraacetic acid (EDTA) at 4°C overnight onto white MicroFluor plates (Thermo Scientific, Rockford, IL, USA). The plates were washed with PBS-EDTA three times using an automated plate washer and blocked with PBS-EDTA containing 0.5% fish gelatin at room temperature for 1 h. Plasma samples were diluted and measured in triplicate. Antibody binding was detected with an appropriate alkaline phosphatase-labeled goat anti-mouse secondary antibody [Sigma-Aldrich, goat anti-mouse IgM (μ-chain specific)-alkaline phosphatase, goat anti-mouse IgG (Fc specific)-alkaline phosphatase, and goat anti-mouse IgA (α-chain specific)-alkaline phosphatase]. Chemiluminescence was detected using LumiPhos530 substrate (Lumigen Inc. Southfield, MI, USA) with Wallac Victor³ multilabel reader, and expressed as relative light units (RLU) measured in 100 milliseconds (ms).