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

Author:

Topbas Celalettin1,Swick Alan1,Razavi Morteza2,Anderson N Leigh2,Pearson Terry W2,Bystrom Cory1

Affiliation:

1. Cleveland HeartLab, Cleveland, OH

2. SISCAPA Assay Technologies, Inc., Washington, DC

Abstract

Abstract BACKGROUND Lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme associated with inflammation, is used as a biomarker for cardiovascular disease risk. Both the concentration and activity of Lp-PLA2 have been shown to be clinically relevant. However, there is a discordance between the serum concentration of Lp-PLA2 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-PLA2 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-PLA2 and lipoproteins and the effect of selected detergents in liberating Lp-PLA2 were evaluated by use of immunoprecipitation and Western blot analyses. RESULTS Serum Lp-PLA2 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-PLA2 from lipoprotein complexes dramatically increased the amount of protein detected by immunoassay and improved the agreement with activity measurements. CONCLUSIONS Quantitative analysis of Lp-PLA2 concentration and activity by LC-MS/MS assays provided key insight into resolving the well-documented discordance between Lp-PLA2 concentration (determined by immunoassay) and activity. Quantitative detection of Lp-PLA2 by immunoassay appears to be strongly inhibited by interaction of Lp-PLA2 with lipoprotein. Together, the results illustrate the advantages of quantitative LC-MS/MS for measurement of Lp-PLA2 concentration (by SISCAPA) and activity (by direct product detection).

Publisher

Oxford University Press (OUP)

Subject

Biochemistry, medical,Clinical Biochemistry

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5. Role of Lipoprotein-Associated Phospholipase A2 in Atherosclerosis;Zalewski;Arterioscler Thromb Vasc Biol,2005

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