Lp-PLA2 Overview
Summary and Explanation
Lp-PLA2 is a calcium-independent serine lipase that is associated with low-density lipoprotein (LDL) in human plasma and serum [1] and is distinct from other phospholipases such as cPLA2 and sPLA2 [2]. Lp-PLA2 is produced by macrophages and is expressed in greater concentrations in atherosclerotic lesions [3]. Several lines of evidence suggest that oxidation of LDL plays a critical step in the development and progression of atherosclerosis [4,5]. Lp-PLA2 participates in the oxidative modification of LDL by hydrolyzing oxidized phosphatidylcholine generating lysophosphatidylcholine and oxidized free fatty acids, both of which are potent proinflammatory products that contribute to the formation of atherosclerotic plaques [6,7,8]. Lp-PLA2 has demonstrated modest intra- and inter-individual variation,commensurate with other cardiovascular lipid markers and substantially less than C-reactive protein(CRP). In addition, Lp-PLA2 is not elevated in systemic inflammatory conditions, and may be a more specific marker of vascular inflammation. The relatively small biological variation of Lp-PLA2 and its specificity are of value in the detection and monitoring of cardiovascular risk [9].
Elevated levels of Lp-PLA2, as measured by immunoassay, were found in patients with angiographically proven coronary heart disease (CHD), when compared to age matched controls [1]. In a retrospective case-control study, using samples from hypercholesterolemic men (n=6595) in the West of Scotland Coronary Prevention Study (WOSCOPS), a 2-fold greater risk of coronary heart disease was observed for subjects in the highest quintile of Lp-PLA2 levels, compared to the lowest quintile. Furthermore, the CHD risk association of Lp-PLA2 was shown to be independent of LDL and other markers of inflammation: C-reactive protein, white cell count and fibrinogen. The authors of the study stated in their conclusions, “Elevated levels of lipoprotein-associated phospholipase A2 appear to be a strong risk factor for coronary heart disease, a finding that has implications for atherogenesis and the assessment of risk” [10]. In another report, using samples from the Atherosclerosis Risk in Communities (ARIC) study, which followed 12,819 apparently healthy middle-aged (45–64 years) men and women for six to eight years, Lp-PLA2 was found to be an important predictor of CHD risk. For individuals with LDL less than 130 mg/dL, Lp-PLA2 was significantly and independently associated with a 2-fold higher risk for CHD events including the need for revascularization, myocardial infarction and death from cardiac disease [11].
The ARIC study was re-analyzed to determine the risk of stroke associated with increased levels of Lp-PLA2. A total of 223 stroke events were identified from the study group; of this, 194 (87%) were ischemic stroke associated with atherosclerosis, as classified by the ARIC investigators. This proportion of ischemic stroke to the total is consistent with the percentage found in the general population [12]. The results of this study indicated that Lp-PLA2 was a strong predictor of these strokes, with an increased risk of nearly 2-fold, even after adjustment for blood pressure, lipids, diabetes, body mass index and other inflammatory markers [13].
Turbidimetric Immunoassay Format
Intended Use
The PLAC® Test Reagent Kit is a turbidimetric immunoassay for the quantitative determination of Lp-PLA2 (lipoprotein-associated phospholipase A2) in human plasma or serum on automated clinical chemistry analyzers, to be used in conjunction with clinical evaluation and patient risk assessment as an aid in predicting risk for coronary heart disease, and ischemic stroke associated with atherosclerosis.
Principle of the Test
The PLAC Test is a turbidimetric immunoassay using two highly specific monoclonal antibodies (2C10 and 4B4) for the direct measurement of Lp-PLA2 concentration in human plasma or serum [14]. Lp-PLA2 in patient samples binds specifically to the monoclonal antibodies that are linked to polymeric microparticles in suspension. As Lp-PLA2 binds, a change in suspension turbidity occurs, resulting in a measurable absorbance change that is read at 570 nm on a clinical chemistry analyzer. This change in absorbance is proportional to the concentration of Lp-PLA2 in the patient sample. A set of Lp-PLA2 calibrators is used to plot a standard curve of absorbance versus Lp-PLA2 concentration from which the Lp-PLA2 concentration in the test sample can be determined by interpolation.
ELISA Format
Intended Use
The diaDexus PLAC® test is an enzyme immunoassay for the quantitative determination of Lp-PLA2 (lipoprotein-associated phospholipase A2) in human plasma and serum, to be used in conjunction with clinical evaluation and patient risk assessment as an aid in predicting risk for coronary heart disease, and ischemic stroke associated with atherosclerosis.
Principle of the Test
The diaDexus PLAC Test is based on the principle of a sandwich enzyme immunoassay using two specific monoclonal antibodies described by Caslake et al. [1]. The assay system utilizes monoclonal anti-Lp-PLA2 antibodies (2C10) directed against Lp-PLA2 for solid phase immobilization on the microwell strips. Sample is added to the plate and incubated for 10 minutes at 20–26 °C. A second monoclonal anti-Lp-PLA2 antibody (4B4) labeled with the enzyme horseradish peroxidase (HRP) is then added and reacted with the immobilized antigen at 20–26 °C for 180 minutes, resulting in the Lp-PLA2 molecules being captured between the solid phase and the enzyme-labeled antibodies. The wells are washed with a supplied buffer to remove any unbound antigen. The substrate, tetramethylbenzidine (TMB), is then added and incubated at 20–26 °C for 20 minutes, resulting in the development of a blue color. Color development is stopped with the addition of Stop Solution, changing the color to yellow. The absorbance of the enzymatic turnover of the substrate is determined spectrophotometrically at 450 nm and is directly proportional to the concentration of Lp-PLA2 present. A set of Lp-PLA2 Calibrators is used to plot a standard curve of absorbance versus Lp-PLA2 concentration from which the Lp-PLA2 concentration in the test sample can be determined.