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 1020 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).
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