For the quantitative determination of human Phospholipase A2 Group VII (PLA2G7) concentrations in cell culture supernates, serum, and plasma.
(Precision within an assay)
Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.Inter-assay Precision
(Precision between assays)
Three samples of known concentration were tested in twenty separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components.
Twenty assays were evaluated and the minimum detectable dose (MDD) for human PLA2G7 ranged from 0.025-0.284 ng/mL. The mean MDD was 0.074 ng/mL.
The MDD was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.
Phospholipase A2 Group VII (PLA2G7), also known as lipoprotein-associated phospholipase (Lp-PLA2) and platelet-activating factor acetylhydrolase (PAF-AH), is a secreted 55-67 kDa calcium-independent glycoprotein that belongs to the phospholipase A2 superfamily (1-3). The active site of the 420 amino acid mature human PLA2G7 is composed of a serine, histidine, and aspartic acid triad, which catalyzes the hydrolysis of the sn-2 ester bond of phospholipids (4-6). Substrates of PLA2G7 are limited to PAF (1-O-alkyl-2-acetyl-snglycero-3-phosphocholine), a potent pro-inflammatory phospholipid mediator, and PAF-like molecules that are generated upon oxidation of phospholipids on low density lipoprotein (LDL) particles (2,7,8). PLA2G7 is produced mainly by hematopoietic cells such as monocytes/ macrophages, lymphocytes, mast cells, megakaryocytes and platelets (9). Secreted PLA2G7 contains asparagine-conjugated carbohydrate containing sialic acid residues, which prevents association of the enzyme with high density lipoprotein (HDL) particles. In humans, circulating PLA2G7 is mainly bound to LDL (80%), a smaller amount is also bound to HDL (20%) (2, 10-12).
PLA2G7 activity has been suggested to have a dual role in atherogenesis. Because it degrades and inactivates PAF and PAF-like pro-inflammatory phospholipid mediators, it is suggested to play an anti-atherogenic/anti-inflammatory role (2, 7, 8). However, LDL bound PLA2G7 also has a crucial role in the formation of atherosclerotic plaques (9, 11, 13-16). When LDL is trapped in the subendothelial space at the site of new plaque formation (as a result of the interaction between its basic apolipoprotein B100 molecules and the negatively-charged subendothelial proteoglycans), the associated lipids are subjected to oxidative modifications. LDL associated PLA2G7 then hydrolyzes the oxidized phospholipids to release lysophosphatidylcholine and oxidized non-esterified fatty acid, both of which are pro-inflammatory and can stimulate the production of cytokines, upregulate adhesion molecules, recruit macrophages, and ultimately lead to foam cell formation. The balance between the pro- and anti-inflammatory roles of PLA2G7 may depend on the concentration of the available substrates. In humans, multiple recent studies have pointed to a pro-atherogenic role of PLA2G7. Increased plasma levels of the enzyme have been shown to be a good predictor of cardiovascular disease and have a strong association with atherosclerosis (17-20).
The Quantikine Human PLA2G7/PAF-AH/Lp-PLA2 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human PLA2G7 in cell culture supernates, serum, and plasma. It contains E. coli-expressed recombinant human PLA2G7 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human PLA2G7 showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring human PLA2G7.