BIOXYTECH pl·GPx-EIA Enzyme Immunoassay for Human Plasma Glutathione Peroxidase
Glutathione peroxidases are selenoenzymes which catalyse the reduction of hydroperoxides (H2O2 or ROOH) in the presence of glutathione (GSH). Human blood contains, in addition to Se-GPx in erythrocytes, a plasma-specific glutathione peroxidase (pl·GPx). pl·GPx is a tetramer of approximately 94-100 kDA. (Maddipati, 1987; Takahashi, 1987). Each of the 4 identical subunits contains an active site with an essential selenocysteine residue. pláGPx differs from the other glutathione peroxidases by its primary sequence, its glycosylated N-terminal region and its extra-cellular location. Initially purified from human plasma, pl·GPx has also been found in human milk (Avissar, 1991). Recently, it was reported that pl·GPx is mostly synthesized and secreted by renal cells from rat kidney (Yoshimura, 1991). Another study has shown that a human liver hepatoblastoma cell line (Hep G2) synthesizes and secretes pl·GPx (Avissar, 1989).
Principle:
The pl·GPx-EIA method is an enzyme-linked immunoassay (ELISA). Samples are incubated in the wells of a sectionable microplate, which have been coated with polyclonal antibodies which are specific for human pl·GPx. These antibodies have been obtained by using a synthetic antigen and have been purified by affinity chromatography (Patent No 9310504).
The presence of pl·GPx is detected by means of a biotinylated-polyclonal antibody to pl·GPx. The final step of the assay is based on an amplification by a biotin-streptavidin coupling in which streptavidin has been covalently linked to alkaline phosphatase. The amount of pl·GPx is enzymatically measured after enzymatic revelation with para-nitrophenyl-phosphate (pNPP). This requires a microplate-reader able to read the absorbance at 405 nm wavelength.
For Research Use Only
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