Quantifying Circulating PON-1 Levels and Activity

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.