Measurement of OxPL-apoB levels OxPL-apoB levels were measured in a chemiluminescent immunoassay using the murine monoclonal antibody E06 that recognizes the phosphocholine (PC) group on oxidized but not on native phospholipids (Taleb et al (1) and references therein). E06 similarly recognizes the PC covalently bound to BSA, as in PC-BSA. A 1:50 dilution of plasma in 1% BSA in TBS was added to microtiter wells coated with the apoB-100 specific monoclonal antibody MB47, which binds a saturating amount of apoB-100 to each well, and biotinylated E06 was then used to determine the content of OxPL-apoB. These values are reported as nanomolar (nM) PC-OxPL using a standard curve of nM phosphocholine (PC) equivalents, as recently described (2). Because each well contains equal numbers of apoB-100 particles, the OxPL-apoB value reflects the absolute content of OxPL per a constant amount of captured apoB lipoprotein. It thus represents an OxPL-apoB value that is independent of plasma levels of apoB-100 or of LDL cholesterol. Furthermore, the assay detects only the subset of OxPL detected by antibody E06 and not all species of OxPL.The OxPL-apoB assay has historically taken 3 modifications/optimizations to allow easier translation to the bedside. The assay was set up a priori to quantitate the amount of OxPL bound to apoB-containing lipoproteins captured on microtiter well plates (3). It was thus called OxPL/apoB (E06) and initially represented a true ratio by performing 2 measurements- the OxPL measure in one set of plates and the apoB captured on the plate in a second set of plates (we emphasize that the denominator is not the plasma level of apoB but reflects how much apoB is captured on the plates). The ratio of OxPL/apoB was then determined and is a unitless measure. In this first version of the assay, an anti-apoB antibody (MB47) that recognizes all apoB-containing lipoproteins was used to capture a saturating amount of apoB from a 1:50 dilution of plasma sample. The amount of apoB captured on the plate was then quantitated with a second anti-apoB antibody and the results expressed in relative light units (RLUs). This measure represented the denominator of the OxPL-apoB ratio. In parallel, a second set of plates was coated with MB47 and then the OxPL content on apoB measured with monoclonal antibody E06 and results expressed as RLUs. E06 binds to the phosphocholine (PC) headgroup of oxidized but not normal phospholipids. The ratio OxPL/apoB was then determined. This assay was designed purposefully this way to make it independent of plasma apoB and LDL-C levels. With this methodology, all wells capture the same amount of apoB at plasma apoB levels of 10-20 mg/dl. Virtually all patients have a higher concentration of apoB than 10-20 mg/dl, therefore, all plates would be expected to be equally saturated with apoB. A second version of this assay was created in 2006 to reduce the time and expense of running both assays to determine OxPL/apoB ratio (4). We determined in larger datasets that assays measuring microtiter well content of apoB were not needed, and started to report the OxPL/apoB assay as OxPL/apoB (E06/MB47) in RLUs, i.e. not determining the apoB content of plates. The rationale for this is that after showing all plates were equally saturated with apoB, we showed that the correlation between OxPL/apoB ratio and OxPL-apoB RLUs was >0.995 with similar results on prospective CVD outcomes. Therefore, we deleted this step in future studies.A third version was developed in 2012 (2), which is the current version in the TNT paper, we further generated a standard curve by plating phosphocholine-modified bovine serum albumin (PC-BSA) with known amounts of PC as the standard and determining E06 immunoreactivity in RLUs (2). E06 recognizes the PC present on BSA similar to the PC in OxPL. Because each mole of OxPL contains one mole of PC, using the RLUs from PC-BSA standards, we can convert the RLUs of the E06/MB47 measures to OxPL equivalents. Thus, we now convert the RLUs to nanomoles of PC-OxPL and report this as nanoMolar (nM) units PC-OxPL. This measure reflects the absolute content of OxPL and is not a ratio. The subset of OxPL recognized by E06, and not all OxPL, but we never claimed otherwise and stated plainly in the methods that these OxPL, which represent a variety of species as initially defined by Friedman et al (5) as measured by antibody E06.In prior studies (1,6-9), this variable was expressed as OxPL/apoB, reflecting the fact that this measure quantitates the number of OxPL moles per unit mass of apolipoprotein B-100 present on microtiter well plates (and not the level in the circulation). The nomenclature is now changed to OxPL-apoB to minimize confusion that this measure represents a ratio of OxPL divided by plasma levels of apoB. Within-person 5-year reproducibility of frozen samples has been shown to be high (r=0.78) (4) and pilot-tests showed that OxPL-apoB levels are stable over 24 hours on ice (intraclass correlation coefficient 0.96)(2) as well as frozen samples stored under long term conditions (4,10-12).

References

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