Submitted on August 6, 2007
Revised on September 11, 2007
Accepted on September 27, 2007
Development of an immunoblot assay with infrared fluorescence to quantify paraoxonase 1 in serum and plasma
Philip W. Connelly, Graham F. Maguire, Clive M. Picardo, John F. Teiber, and Dragomir Draganov
J. Alick Little Lipid Research Laboratory, St. Michael's Hospital, Toronto, ON M5B 1W8
Corresponding Author: connellyp{at}smh.toronto.on.ca
Paraoxonase 1 (PON1) requires calcium for activity and is inactivated in the presence of EDTA. Because of this, studies to date have used serum or heparinized plasma for both activity and mass assays of PON1. Whole serum and EDTA plasma were analysed by SDS electrophoresis and Western blot using anti-paraoxonase 1 monoclonal antibody 4C10. Because PON1 has one disulfide and one free cysteine residue, the samples were reduced with dithiothreitol prior to electrophoresis. Western blot identified a major paraoxonase 1 band with a molecular weight of ~45 kD and two minor bands of about 40 and 35 kD in both serum and EDTA plasma. This established that PON1 is inactive, but structurally intact, in EDTA plasma and suggested that a mass assay could be developed based on SDS-electrophoresis and Western blot. Linearity was established for plasma and for a PON1 standard. Quantification was based on the major PON1 band at 45 kD. The correlation between serum and plasma PON1 mass was 0.9553. The between run variation was determined with a serum pool to be 6%. The mass of PON1 in serum was significantly correlated with arylesterase activity (r= 0.85). Thus we have demonstrated the feasibility of measuring PON1 mass in either serum or EDTA plasma.
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