Substrate solution

VEGF concentration in conditioned medium was determined using the human VEGF DuoSet ELISA Development kit (R&D Systems), according to the instructions of the manufacturer. In short, the plate was coated with a capture antibody against VEGF followed by blocking of the wells with reagent diluent (PBS with 1% BSA). Next, samples were added to the plate and bound VEGF was detected with biotinylated detection antibody, followed by addition of streptavidin conjugated to HRP. In between each step the plate was washed 3 times with wash buffer (PBS with 0.05% Tween-20). Substrate solution (R&D Systems) was added to the plate, reaction was stopped with 2 N sulfuric acid (H₂SO₄) and the optical densities of the wells were determined at 450 nm on a Synergy HT microplate reader from BioTek, with wavelength correction set to 540 nm. The amount of VEGF in the samples was quantitatively determined according to a 7-point standard curve of 2× serial dilutions of known concentrations. Both standards and samples were measured in triplicates.

The TGF-β1 content of conditioned medium was determined using the DuoSet ELISA for human TGF-β1 (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions. Briefly, wells were coated with mouse anti-TGF-β1 (200 ng in PBS) and incubated for 18 h. The wells were washed with 0.05% Tween 20 in PBS, blocked with 5% Tween 20 in PBS for 1 h, and incubated with conditioned medium (100 µL) for 2 h. Control wells were incubated with fresh medium. For standard curves, wells were incubated with TGF-β1 (100 µL) (31.3 to 2000 pg/mL) in 1.4% BSA in PBS. With washing between steps, wells were incubated with 100 µL of biotinylated chicken anti-human TGF-β1, (300 ng/mL) for 2 h, then streptavidin-horseradish peroxidase (100 µL) for 20 min, followed by 100 µL Substrate Solution (R&D Systems) for 20 min, in reduced lighting. The reaction was stopped with 50 µL 1 M H₂SO₄, and absorbance (450 nm) was measured using a Titertek Multiscan microplate reader (Flow Laboratories).

Circulating levels of human total PON-1 were measured in Lithium-heparin plasma by an enzyme-linked immunosorbent assay (ELISA) purchased from R&D Systems (catalog No. DYC5816-5) and performed according to the manufacturer’s recommendations. Samples for ELISA were prepared at 100× dilution in sample diluent purchased from R&D Systems (catalog No. DYC001). The ELISA assay kit contained human total PON-1 capture antibody, detection antibody, PON-1 standard and streptavidin HRP. Additional reagents such as reagent diluent (catalog No. DY995), substrate solution (catalogue No. DY999), and stop solution (catalog No. DY994) were also purchased from R&D systems. The minimum and maximum amount of detectable PON-1 were 0.15 ng/mL and 10 ng/mL, respectively. Western blot was performed independently using a monoclonal antibody to PON-1 [31 (link)] to validate the ability of the ELISA to detect the presence of circulating PON-1 protein in plasma (Figure S4).
Circulating lactonase activity of PON was measured in the patient serum samples with a commercially available fluorometric assay (BioVision Incorporated, catalog # K999-100). Serum PON lactonase activity was calculated as the hydrolytic activity toward a fluorogenic benzopyran-2-one substrate of PON in the presence and absence of a specific PON inhibitor (2-hydroxyquinoline) according to the manufacturer’s protocol.

Fibroblasts were seeded at a density of 31,250 cells cm^− 2 in 96 well plates. After attachment overnight, they were rested for 72 h in DMEM + 1 v/v% FCS before treatment with conditioned medium for 24 h. The media were collected to measure VEGF and the cell monolayer was washed in PBS before lysis for a protein assay. 96 well plates were coated in mouse anti-human VEGF capture antibody and blocked in 2 w/v% BSA in PBS before addition of 100 μl of samples and VEGF standards of 0–10,000 pg ml^− 1 and incubation overnight at 4 °C. The medium was removed, wells washed in 0.05 v/v% tween/PBS, and a detection antibody (biotinylated goat anti-human VEGF) was added for 1 h at room temperature. This was removed and wells incubated with Streptavidin-HRP at a 1:400 dilution for 1 h. After a final wash, 100 μl of substrate solution (R&D) was added per well and incubated for approximately 30 min or until blue coloration was seen in the standard wells. 100 μl of 2 N H₂SO₄ was added to stop the reaction and the absorbance read at 450 nm.