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Clin Chem. 2018 Apr;64(4):697-704. doi: 10.1373/clinchem.2017.279752. Epub 2018 Jan 10.

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

Author information

1
Cleveland HeartLab, Cleveland, OH.
2
SISCAPA Assay Technologies, Inc., Washington, DC.
3
Cleveland HeartLab, Cleveland, OH; cbystrom@clevelandheartlab.com.

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).

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