Measurement of Lipoprotein-Associated Phospholipase A2 by Use of 3 Different Methods: Exploration of Discordance between ELISA and Activity Assays

BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA(2)), an enzyme associated with inflammation, is used as a biomarker for cardiovascular disease risk. Both the concentration and activity of Lp-PLA(2) have been shown to be clinically relevant. However, there is a discordance between the serum concentration of Lp-PLA(2) measured by the standard ELISA-based immunoassays and the activity of this enzyme, leading to substantial discordance in risk categorization depending on assay format. METHODS: We developed 2 LC-MS/MS-based assays to quantify serum Lp-PLA(2) activity (multiple reaction monitoring detection of product) and concentration [stable isotope standards and capture by antipeptide antibody (SISCAPA) immunoaffinity], and we investigated their correlation to commercially offered colorimetric activity and immunometric concentrations assays. Associations between Lp-PLA(2) and lipoproteins and the effect of selected detergents in liberating Lp-PLA(2) were evaluated by use of immunoprecipitation and Western blot analyses. RESULTS: Serum Lp-PLA(2) concentrations measured by quantitative SISCAPA-mass spectrometry were substantially higher than concentrations typically measured by immunoassay and showed an improved agreement with Lp-PLA2 activity. With detergents, liberation of Lp-PLA(2) from lipoprotein complexes dramatically increased the amount of protein detected by immunoassay and improved the agreement with activity measurements. CONCLUSIONS: Quantitative analysis of Lp-PLA(2) concentration and activity by LC-MS/MS assays provided key insight into resolving the well-documented discordance between Lp-PLA(2) concentration (determined by immunoassay) and activity. Quantitative detection of Lp-PLA2 by immunoassay appears to be strongly inhibited by interaction of Lp-PLA(2) with lipoprotein. Together, the results illustrate the advantages of quantitative LC-MS/MS for measurement of Lp-PLA(2) concentration (by SISCAPA) and activity (by direct product detection). (c) 2017 American Association for Clinical Chemistry