Glutathione Peroxidase (GPx) is a family of proteins that catalyze the reduction of hydrogen peroxides and organic hydroperoxides by glutathione. Several isoforms exist that differ in their primary structure and location. The classical cytosolic/mitochondrial Gpx1 (cGpx) is selenium dependent and the first of the Gpx family to be discovered. A gastrointestinal form (GiGpx or GPX2), is an intracellular enzyme expressed only in GI system epithelial cells. An extra-cellular form (plGpx or GPx3) is mainly expressed by the kidney where it is released into the blood circulation. Phospholipid hydroperoxide (PhGpx or GPx4) expressed in most tissues, can reduce many hydroperoxides including those integrated into membranes and hydroperoxy lipids in low density lipoprotein or thymine. Mammalian GPx family members, except for the recently described Cys containing GPx3 and epididymis-specific secretory GPx (eGpx or GPx5), possess selenocysteine at the active site.
The NWLSS™ cGPx Assay is based on a sandwich Enzyme-Linked Immunosorbent Assay (ELISA). The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific to human cGPx. This stationary phase antibody binds sample or standard cGPx while nonbound proteins are removed by washing. Next, bound cGPx is tagged with a biotin-conjugated monoclonal antibody specific for MnSOD followed by Avidin conjugated to Horseradish Peroxidase (HRP). Subsequent addition of TMB-substrate solution causes blue color (650 nm) development proportional to the amount of cGPx originally captured by the stationary phase antibody. Finally, addition of a sulfuric acid solution stops the reaction resulting in a yellow color product measured at 450 nm. Sample cGPx concentration is determined by comparing the 450 nm absorbance of sample wells to the absorbance of known standards.