CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. provisional patent application no. 61/893, 1 14, filed on 10/18/2013, titled "METHOD FOR DETECTION OF LIPOPROTEIN- SPECIFIC Lp-PLA2 ASSOCIATION", herein incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

FIELD

[0003] Described herein are compositions, kits and methods related to detection for detection of specified lipoproteins expressing Lp-PLA2 in a sample.

BACKGROUND

[0004] Several lines of evidence suggest that the role of plasma Lp-PLA ₂ (lipoprotein- associated phospholipase A2) in atherosclerosis may depend on the type of lipoprotein particle with which this enzyme is associated. Although data from large population studies have shown an independent association between the plasma Lp-PLA ₂ levels and the risk of a future cardiovascular event, it is generally believed that only about 70-80% of Lp-PLA ₂ is LDL- associated Lp-PLA ₂ . Lp-PLA ₂ is also known to associate with other lipoproteins, including HDL. Further, several lines of evidence suggest that HDL-associated Lp-PLA ₂ may substantially contribute to HDL's anti-atherogenic activities. In addition, there is also evidence that oxidized phospholipids are preferentially sequestered on the phospholipid Lp(a) (an LDL- like lipoprotein) before being metabolized by Lp(a)-associated Lp-PLA . Taken together, it is generally thought the distribution of Lp-PLA ₂ among the different lipoproteins may result in different patient outcomes.

[0005] Unfortunately, to date there has not been a reliable assay for determining the association of Lp-PLA ₂ and particular lipoproteins/subclasses of lipoproteins.

[0006] For example, work from the Tsimikas laboratory described a prototype assay in which the analyte levels of Lp-PLA ₂ associated with specific lipoproteins were quantified with a hybrid ELISA-based approach using antibodies against different target antigens. The hybrid ELISA described by Tsimikas and colleagues has an antibody pair with specificities directed against both an individual lipoprotein antigen and an Lp-PLA ₂ antigen. For example, Arai et al. (J Lipid Res. 2012 August; 53(8): 1670-1678 (2012), summarized in FIG. 1 A) used a configuration where individual antibodies recognizing three different individual lipoprotein subspecies were used as the capture antibody in combination with a common detection antibody recognizing Lp-PLA ₂ (i.e., a 4B4-HRP conjugate available from diaDexus). Efforts to replicate the methods of Arai et al. using conceptually similar reagents in an analogous assay

configuration (see below, in reference to FIG. IB) have been entirely unsuccessful. One potential explanation for the difficulties experienced pertains to the sub-stoichiometric relationship of Lp-PLA ₂ with the individual lipoproteins, which may be between 1 : 100 and 1 : 10,000. A significant technical concern is that the immobilized lipoprotein-specific coating antibody may become saturated with non-Lp-PLA ₂ -containing lipoprotein complexes, resulting in a low signal generation with the a-Lp-PLA ₂ detection antibody when used with a wide variety of actual patient samples.

[0007] Described herein are methods and systems capable of achieving adequate signal in an assay (e.g., a hybrid ELISA assay) using a PLAC antibody (Lp-PLA ₂ antibody). These methods and systems may allow determination of lipoprotein specific Lp-PLA ₂ levels in patient samples and/or the determination of relative levels of Lp-PLA ₂ associated with different lipoproteins.

SUMMARY OF THE DISCLOSURE

[0008] Here, we describe immunoassays that provide a sensitive and highly reproducible assay to determine lipoprotein specific Lp-PLA ₂ levels in patient samples and/or the relative levels of Lp-PLA ₂ associated with different lipoproteins. Described herein are assay

configurations in which an antibody recognizing an Lp-PLA ₂ antigen was used as the capture antibody, which may be bound to a substrate, and one or a set of individual antibody conjugates recognizing various lipoprotein subspecies (for example, Apo-Al, Apo-B and apo[a]) are used as a detection antibody. In some variations the subspecies may be assayed in parallel, either separately or concurrently. Antibodies may be conjugated to different reporters (e.g., fluorophore).

[0009] In general, the methods and systems implementing these methods includes the use of a Lp-PLA2 antibody as the capture antibody to initially capture lipoproteins associated with Lp- PLA2, then screening the resulting enriched population for specific lipoprotein subclasses (e.g., LDL, HDL, Lp(a)). This approach is the inverse of prior art systems, in which capture antibodies were directed to lipoproteins which were then screened to determine association with Lp-PLA2. [00010J For example, described herein are methods of detecting lipoprotein particles having an associated Lipoprotein-associated phospholipase A2 (Lp-PLA2) molecule from a patient sample. A method may include: enriching a patient sample for lipoproteins expressing Lp- PLA2; and detecting a subclass of lipoprotein particles from the enriched sample. Enrichment of the patient sample typically refers to enrichment by binding of Lp-PLA2 within the patient sample to a solid-phase material to which a capture molecule for Lp-PLA2 has been linked.

[00011] A method of detecting lipoprotein particles having an associated Lipoprotein- associated phospholipase A2 (Lp-PLA2) molecule from a patient sample may include, for example: exposing a solid-phase support material in which a capture molecule that specifically binds Lp-PLA2 have been immobilized to the patient sample; and probing the solid-phase support material with a detection molecule specific a subclass of lipoprotein particles.

[00012] Any of these methods may also be methods of detecting a patient's relative association of Lipoprotein-associated phospholipase A2 (Lp-PLA2) with one or more lipoproteins. For example, the method may include: exposing a solid-phase support material to a patient sample, wherein a capture molecule that specifically binds Lp-PLA2 has been immobilized on the one or more solid phase supports; probing the solid-phase support material with a detection molecule specific to Lp-PLA2; and probing the solid-phase support material with a detection molecule specific to a subclass of lipoprotein particles.

[00013] The capture molecule that specifically binds Lp-PLA2 may be, for example, an antibody or antibody fragment directed to Lp-PLA2.

[00014] Any of these methods may include incubating the solid-phase support in the patient sample for greater than 10 minutes before washing the solid-phase support material, to enrich the sample. In any of these variations, probing may comprise probing with a labeled antibody (and/or antibody fragment, e.g., Fab, etc.). Probing the solid-phase support material with a detection molecule specific to Lp-PLA2 and probing the solid-phase support material with a detection molecule specific to a subclass of lipoprotein particles may comprise probing parallel samples including the solid-phase support material. Alternatively, probing the solid-phase support material with a detection molecule specific to Lp-PLA2 and probing the solid-phase support material with a detection molecule specific to a subclass of lipoprotein particles may comprise probing the same sample including the solid-phase support material. Probing may comprises probing with an antibody specific to one or more of: Apo-Al, ApoB-100/-48, and Apo(a). In some variations, the methods include probing the solid phase support with an antibody to a second (or more) subclass of lipoprotein particles, and may also include calculating a ratio from the detected levels of the different subclasses of lipoprotein particles. [00015] In general, any of these methods may also include calculating a ratio from detected levels of the lipoprotein particles and the Lp-PLA2 determined by the probing steps.

[00016] Also described herein are kits for detecting lipoprotein particles having an associated Lipoprotein-associated phospholipase A2 (Lp-PLA2) molecule from a patient sample. A kit may include: a solid-phase support material to which a protein that specifically binds Lp-PLA2 has been immobilized; a wash solution; a buffer solution; a first detection antibody specific to a first subclass of lipoprotein particles; and a second detection antibody specific to a second subclass of lipoprotein particles. BRIEF DESCRIPTION OF THE DRAWINGS

[00017] FIG. 1 A schematically illustrates prior-art assays to determine levels of lipoproteins associated with Lp-PLA ₂ (described, for example, in figure 1C of Ami et al (2012)).

[00018] FIG. IB schematically illustrates a replicated version of a prior art assays to determine levels of lipoproteins associated with Lp-PLA ₂ .

[00019] FIG. 2 shows the results of an ELISA assay using a commercially available

Mercodia Lp(a) ELISA (positive control) with alpha-Apo(a) as both capture and detection antibody from a serum sample.

[00020] FIG. 3 shows the results of an ELISA assay using the commercially available Mercodia Lp(a) as a capture antibody and 4B4 (Lp-PLA2) antibody as a detection antibody from a serum sample.

[00021] FIG. 4 shows the results of an ELISA assay using 2C 10 as a capture antibody and Mercodia Lp(a) as a detection antibody from a serum sample.

[00022] FIG. 5 schematically illustrates the configuration for detection of lipoprotein- associated Lp-PLA2 described herein.

[00023] FIG. 6 shows the results of an ELISA assay using an Lp-PLA2 capture antibody (2C10) with a variety of different detection antibodies targeting specific subclasses of lipoproteins, including LDL, Lp(a), and HDL from a serum sample.

[00024] FIGS. 7A-7C show graphs analyzing the Lp-PLA2 (e.g., 2C10 antibody) capture and enrichment method for various lipoprotein detection antibody configurations.

[00025] FIG. 8A is a schematic illustration of one variation of a method for detecting subclasses of lipoproteins (such as HDL, LDL, etc.) that associate with Lp-PLA2 form a patient sample.

[00026] FIG. 8B is a schematic illustration of different methods (method 1, method 2) for detecting subclasses of lipoproteins that associate with Lp-PLA2 from a patient sample. [00027] FIG. 9 is a comparison of the two methods for detecting subclasses of lipoproteins illustrated in FIGS. 8B using patient samples. In this example, method 2 resulted in a slightly higher signal, which may represent an enrichment of the Lp-PLA2 and associated lipoprotein.

[00028] FIGS. 10A and 10B illustrate the results of a side-by-side comparison of an Lp-PLA2 capture method (similar to that shown in FIGS. 8A and 8B, also referred to as a "Hybrid ELISA" assay) and a prior art Lp-PLA2 detection assay (the commercially available PLAC assay described herein). Although the concentrations of LpPLA2-liproprotein (e.g., ApoB) in this example cannot be directly quantified in this example without a calibrator, the results indicate the detection of Lp-PLA2 and the ability to correlate the results with the patient's physiology.

DETAILED DESCRIPTION

[00029] Described herein are methods and systems (including kits) for detecting lipoproteins, e.g., lipoprotein sub-classes, associated with Lp-PLA2 from a patient sample (material). These systems and methods may initially enrich for Lp-PLA2 containing lipoproteins from a patient material (e.g., blood, blood fraction, bodily fluid, etc.), and then probe the resulting enriched sample to identify one or more (e.g., two, three, four, five, six, etc.) types of lipoproteins, including (but not limited to) HDL, LDL, and Lp(a).

[00030] For example, in the methods, kits, and compositions for determining an association (or fraction) of lipoproteins associated with Lp-PLA2, the methods and systems implementing them may normalize the amount of starting antigen bound in a first incubation. For example, in the first incubation, one may want to bind a significant amount (e.g. virtually all) of the antigen present in the sample so that the sensitivity of the assay is governed significantly (e.g. solely) by the concentration of the detection antibody. The configuration described herein may allow that scenario to be achieved, by using the Lp-PLA2 antibody as a capture antibody to enrich the antigen present.

[00031] In contrast, prior art methods and system described only hybrid assays that use antibodies (which may have different affinities) to capture lipoproteins that are present at different relative concentrations. Although this approach, described in FIGS. 1A and IB, is conceptually straightforward, it has proven difficult to replicate, resulting in very low signals.

[00032] In contrast, the approach described herein may normalize the assay by capturing Lp- PLA ₂ in a first incubation and allowing the lipoprotein-specific detection antibody concentration to be adjusted in a second incubation, thereby potentially allowing calibration to some established concentration of the individual lipoprotein. In addition, configurations described herein may be used as part of a kit including standard reagents and processes to provide novel assays for determining, for example, the distribution of Lp-PLA2 between one or more lipoprotein fractions within a tissue (e.g., blood) sample.

[00033] In particular described herein are novel and surprisingly effective method and systems (including kits) for detecting the association (including in some variations, one or more of the level, amount, percent, degree, fraction, or some combination thereof) of association of Lp-PLA2 with one or more lipoprotein (e.g., LDL, HDL, etc.) and/or with lipoprotein-associated proteins (including sub-classes of lipoproteins). This information may be used by a physician to guide treatment and/or for monitoring of patient health in an acute and/or long-term treatment.

[00034] As described in FIGS. 1 A and IB, prior art techniques probed for the presence of Lp- PLA2 in a population of lipoproteins by first capturing one or more sub-classes of lipoprotein (e.g., ApoB-100 containing lipoproteins, Apo(a) containing lipoproteins, and Apo-Al containing lipoproteins), then assaying with a detection antibody to Lp-PLA2 (using the 4B4 antibody). This assay has proven difficult to reliable replicate and adapt as a clinical assay, as shown by FIG. 3, discussed in greater detail below. Replicating these prior art assays using conceptually similar reagents in an analogous assay configuration has proven challenging. This may be because the sub-stoichiometric relationship of Lp-PLA ₂ with the individual lipoprotein is low (e.g., between 1 : 100 and 1 : 10,000). Further, immobilized lipoprotein-specific coating antibody may become saturated with non-Lp-PLA ₂ -containing lipoprotein complexes, resulting in low signal generation with the a-Lp-PLA ₂ detection antibody.

[00035] The methods and assays (including systems) described herein may provide a solution that is not effected by the stoichiometric relationship of Lp-PLA2 and various lipoproteins. Instead, these assays may begin by enriching for lipoproteins displaying Lp-PLA2 and then probe to determine the presence, and/or distribution of different lipoprotein sub-classes.

Although enrichment as described herein may potentially distort the absolute concentrations of lipoproteins, preliminary work suggests that the distribution of different Lp-PLA2 containing lipoproteins may be clinically useful, and the methods described herein may preserve and reflect the distributions of various Lp-PLA2 binding lipoproteins in a patient sample.

[00036] For example, described herein are assay configurations in which a capture molecule (e.g., an antibody, e.g., 2C10 antibody, diaDexus) recognizing an Lp-PLA ₂ antigen is used as the capture molecule (e.g., antibody) and a set of individual antibody conjugates recognizing various lipoprotein subspecies (such as, Apo-Al, Apo-B and apo[a]; Table 1) may be used as the detection antibody, either alone or in combination. [00037] Table 1: Relationship between Apolipoproteins:Lipoprotein Particles

[00038] By enriching for lipoproteins containing Lp-PLA2, an enhanced signal may be achieved. The methods described herein may enrich in a manner that is independent of the subclass of lipoprotein, allowing determination of normalized and/or relative distributions of various subclasses of lipoproteins containing Lp-PLA2. Thus, the methods described herein may allow the determination of one or more ratios of one or more sub-classes of lipoproteins containing Lp-PLA2. For example, the methods described herein may allow determination of a ratio of HDL containing Lp-PLA2 to LDL containing Lp-PLA2 (or LDL containing Lp-PLA2 to HDL containing Lp-PLA2), HDL containing Lp-PLA2to Lp(a) containing Lp-PLA2 (or Lp(a) containing Lp-PLA2to HDL containing Lp-PLA2), or LDL containing Lp-PLA2 to Lp(a) containing Lp-PLA2 (or Lp(a) containing Lp-PLA2 to LDL containing Lp-PLA2) or a relative fraction of LP A containing Lp-PLA2:Lp(a) containing Lp-PLA2:HDL containing Lp-PLA2. The measure of lipoprotein containing Lp-PLA2 may be normalized by a normalization factor (e.g., based on total capture Lp-PLA2). In any of the assays described herein, the amount of sample applied may be in excess, so that biding to lipoproteins is not limiting. In some variations the same samples, or parallel samples, may be used to determine the distribution of different associated lipoproteins (and/or sub-classes of lipoproteins) from the enriched Lp-PLA2 in a sample. For example, different detection signals (e.g., different flourophores, etc.) may be used for different lipoproteins in the same sample.

[00039] Thus, unlike prior art approaches, the methods and systems (including kits) described herein are not limited by the Lp-PLA ₂ bound lipoprotein concentration, since associated lipoprotein particles are captured first, followed by detection with the lipoprotein-specific antibody on the Lp-PLA ₂ -enriched population of lipoproteins now immobilized on the solid phase support. FIG. 5 is a schematic of one variation of this method.

[00040] Summarized below in Table 2 are descriptions of assays, including a control assay (described in FIG. 2), a replication of prior art assays (described in FIG. 3). The improved assays for detecting lipoproteins associated with Lp-PLA2 from a patient sample described herein were also examined, and are listed in Table 2, and FIG. 6. [00041] Table 2: Summary of Results with Various Hybrid ELISA Configurations Tested

*The apolipoprotein ApoB is not necessarily absolutely specific for LDL, see Table 1 [00042] As described, a hybrid ELISAs, similar to those described by Arai et al. (2012) in which the Hpoprotein-specific antibody was utilized as the coating antibody and a a-Lp-PLA ₂ detection antibody; conjugate, resulted in low signal generation. For example, results using the Mercodia capture antibody-coated plate from their commercially available Lp(a) kit were examined. An identical set of serum samples were run using the kit oc-Apo(a) coated plates coupled with parallel detection performed using both the kit detection antibodyxonjugate (a- Apo[a] -HRP) and the ct-Lp-PLA ₂ detection antibody conjugate (4B4-HRP). Whereas the complete Mercodia Lp(a) ELISA kit gave adequate signal (control, FIG. 2), parallel detection with the Lp-PLA ₂ detection antibodyxonjugate yielded only very weak signal generation (FIG. 3). Conceptually similar configurations in which a Hpoprotein-specific antibody was used as the capture antibody in combination with the 4B4-HRP detection antibody gave weak signal generation (data not shown). These results illustrate that utilizing a capture antibody to bind a specific lipoprotein may result in inadequate signal generation to reliably detect any lipoprotein- associated Lp-PLA ₂ using an antibody to Lp-PLA2 (e.g., the 4B4 detection antibodyxonjugate). In addition, experiments using the 2C 10 capture antibody in combination with the Mercodia ct- apoB conjugate reagent in the Mercodia conjugate buffer also yielded negative results in prototype immunometric assays (FIG. 4).

[00043] Configurations described herein may use a different assay configuration in which a plate or other support substrate (e.g. a 2C10-coated plate such as a standard Gen-3 PLAC ELISA assay) may be used to capture Lp-PLA ₂ antigen, which is followed by detection with lipoprotein- specific polyclonal antibodies directed against a lipoprotein (e.g., ApoB-100, Apo(a) and Apo- A l ). Any appropriate antibody directed to a lipoprotein (monoclonal, polyclonal, fragment, etc.), or to another constituent indicative or characteristic of a lipoprotein particle to be detected, may be used. In the examples described herein (e.g., FIG. 6 and 7), these assays were performed on undiluted human serum samples using otherwise standard PLAC assay conditions (i.e., the standard conjugate diluent, detection antibody incubation times, wash buffers, etc.). Standard PLAC assay conditions are known based on, for example, diaDexus' published PLAC assay. A standard (low) detergent concentration and (high) salt concentration utilized in the PLAC assay may be beneficial to avoid dissociation of Lp-PLA ₂ from the different lipoproteins. Good signal intensity was obtained using all three lipoprotein-specific detection antibodies in these studies (FIG. 6). Of note, different human serum samples demonstrated different OD levels within a given analyte measurement (FIG. 7) and/or different relative OD levels between different analyte measurements on percentage basis (Table 3).

[00044] Table 3: Comparison of OD measurements and Differing Relative Percentages Across Samples

[00045] In general, the lipoprotein-associated phospholipase A2 (Lp-PLA2) enzyme has been shown associate with multiple distinct lipoprotein subspecies, including LDL, HDL and Lp(a), although in "normal" tissue the majority of Lp-PLA2 is believed to be associated with low density lipoprotein particles (LDL), and this combination is thought to be responsible for generating two pro-inflammatory mediators during oxidation of LDL. Apolipoprotein B (ApoB) is the main protein component of LDL and ApoB is regarded as a negative factor in cardiovascular disease. ApoB- 100 is normally 98% of the ApoB in plasma and at a

concentration of approximately 1 mg/ml.

[00046] In some variations, an LpPLA2-Apolipoprotein hybrid Assay as described herein may be used to quantify the Apolipoprotein associated LpPLA2 (or LpPLA2 associated

Apolipoprotein), using a capture molecule (such as an antibody or antibody fragment) that recognizes an Lp-PLA2 antigen (e.g., the diaDexus 2C10 antibody) to capture Lp-PLA2 in a manner that enriches for the capture from the sample, while preserving the association with lipoproteins, and then probing for the lipoproteins or associated markers from the enriched sample. For example, Apo-AI , Apo-B and Lp(a) detection antibodies may be used. In such an enriched system, it may be difficult to quantify the concentration of Lp-PLA2/Apolipoprotein complexes.

[00047] FIG. 8A schematically illustrates one example of a method of detecting lipoprotein particles having an associated Lipoprotein-associated phospholipase A2 (Lp-PLA2) molecule from a patient sample. In this example, an Lp-PLA2 capture antibody (2C10) is bound to a substrate and is exposed to a biological sample (e.g., blood, serum, plasma, etc.). This step may be performed over a sufficient time to drive binding (e.g., 10 min or longer, e.g., 15 min or longer, 20 min or longer, 30 min or longer, 40 min or longer, 1 hr. or longer, etc.) to enrich the patient sample (e.g., the solid phase with binding molecule) with Lp-PLA2, thereby enriching for Lp-PLA2 bound to lipoproteins. Thereafter, the enriched sample (solid phase) may be probed to detect one or more class/subclass of lipoprotein particles. In the examples shown in FIGS. 8A and 8B, the enriched samples are probed for an ApoB protein using an antibody directed to ApoB. Detection may be made, and in some cases quantified, using an indicator such as HRP (e.g., by using a biotinylated detection antibody and a streptavidin-HRP molecule). As illustrated in FIG. 9, this method identified a direct interaction between ApoB and Lp-PLA2 in the LDL (and for these patients, only in LDL).

[00048] FIG. 8B illustrates two alternative methods. In method 1 (of FIG. 8B), an initial incubation to enrich the Lp-PLA2 from the sample by binding to a solid phase substrate (e.g., dish, beads, etc.) onto which anti-Lp-PLA2 was bound at relatively high density. After 10 minutes a lipoprotein detection molecule (e.g., a biotinylated anti-ApoB antibody) was added and the mixture incubated for 3 hours at room temperature before washing multiple times (e.g. 5 times). Alternatively, as shown in the second method, in some variations, the initial step of enriching for Lp-PLA2 on the solid phase substrate may be performed for a longer period (e.g., in this example, 2 hours), followed by a wash. Thereafter, the enriched sample may be probed with the lipoprotein detection moiety, and detected. As illustrated in the comparison between these two methods shown in FIG. 9, the method of enriching for LpPLA2 to the solid phase support/bound LpPLA2 and washing prior to detecting any affiliated molecules (e.g., lipoproteins) enhanced the resulting signals. In this example, the enrichment over >10 min (> 15 min, >20 min, >30 min, >40 min, >1 hr., >1.5 hr., etc.) may allow the LpPLA2 capture molecules (bound anti-Lp-PLA2) to capture more Lp-PLA2 (and therefore more Lp-PLA2/ApoB or other lipoproteins complex).

[00049] In general, the methods described herein may be used to analyze the relative amount of Lp-PLA2 bound to one or more lipoprotein. For example, in some variations the method may identify Lp-PLA2 using an Lp-PLA2 capture molecule and an Lp-PLA2 detection molecule, and in parallel (from parallel or concurrent samples) may identify one or more lipoproteins associated with the Lp-PLA2, as shown in FIGS. 10A and 10B, using an Lp-PLA2 capture molecule and a I ipoprote in-associated detection molecule (e.g., anti-ApoB). In FIGS. 10A and 10B, the relative amounts are determined even in the absence of one or more calibrators for the absolute amounts. FIG. 10B shows a comparison (shown as a percentage of lipoprotein- associated [ApoB] Lp-PLA2 to Lp-PLA2).

[00050] Comparisons of the distribution of Lp-PLA2 among different lipoproteins over time, which may be easily assayed for the first time using the methods and kits described herein, may be used to guide treatment. In particular, within-patient monitoring of the distribution of Lp- PLA2 between one or more lipoproteins (such as ApoB) may be used to guide treatment, indicating the need or benefit of drug or alternative therapies in the prevention and/or treatment of disorders including atherosclerosis, acute and/or chronic inflammation, cardiac disease, coronary heart disease and stroke. Patients that may benefit from such testing and/or monitoring may include those displaying one or more of: moderate risk of cardiovascular disease (>2 risk factors and Framingham 10-year risk score <20%), high risk of cardiovascular disease

(Framingham 10-year risk score >20%), coronary artery disease or risk equivalents (e.g., symptomatic carotid artery disease, peripheral artery disease, abdominal aortic aneurysm, diabetes mellitus), or moderate risk of stroke.

[00051] Although in general the methods described herein are modeled after an ELISA platform to measure (quantitate) proteins associated with Lp-PLA2, other

detection/quantification methods may be used. For example, an enriched sample of Lp-PLA2 may be collected (e.g., by immunoprecipitation as described herein), followed by liquid chromatgrophy/mass spectroscopy (LC-MS), western blotting, or flow cytometry, which may allow both identification of associated lipoproteins as well as quantification. Such methods may also identify non-lipoprotein (including any other proteins/metabolite) that interacts with Lp- PLA2 and may contribute to its clinical/pharmacological/diagnostic utility. For example:

immunoaffinity of Lp-PLA2 as described herein may be followed by mass spectroscopy (MS) analysis to identify associated lipids/metabolites/proteins/peptides that interact in a regulated manner under a variety of health/disease/treatment conditions. Other examples of secondary detection methods that may be used on the enriched Lp-PLA2 samples described herein include ELISAs (direct and competitive, as mentioned above), TIA, Lateral-flow

immunochromatographic assays, microparticle capture, filtration methodologies using

Absorbance, Fluorescence and Chemiluminescence detection principles, or the like.

[00052] As for additional details pertinent to the present invention, materials and

manufacturing techniques may be employed as within the level of those with skill in the relevant art. The same may hold true with respect to method-based aspects of the invention in terms of additional acts commonly or logically employed. Also, it is contemplated that any optional feature of the inventive variations described may be set forth and claimed independently, or in combination with any one or more of the features described herein.

[00053] Although the terms "first" and "second" may be used herein to describe various features/elements, these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

[00054] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word "about" or "approximately," even if the term does not expressly appear. The phrase "about" or

"approximately" may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/- 0.1 % of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), etc. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.

[00055] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others.

Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[00056] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term "invention" merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.