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

Lipoprotein-associated phospholipase A2 (Lp-[PLA.sub.2])3 is an enzyme expressed by macrophages in atherosclerotic plaque (1, 2). In circulation, Lp-[PLA.sub.2] is bound to lipoproteins (3, 4) and functions to hydrolyze oxidized phospholipids, generating products involved in vascular wall inflammation and atherosclerosis (5, 6). Lp-[PLA.sub.2] has been shown to be an independent predictor for coronary heart disease and stroke in multiple studies (7-10).

For clinical use, Lp-[PLA.sub.2] has been assayed by measuring protein concentration or enzyme activity (10-12). Starting in 2003, reference laboratories had access to a Food and Drug Administration (FDA)-approved concentration assay (PLAC[R], diaDexus), commonly referred as the "mass assay." It is a standard indirect antigencapture ELISA that employs 2 monoclonal antibodies, 1 to capture Lp-[LPA.sub.2] from serum and 1 to detect it. In 2015, an FDA-approved Lp-[PLA.sub.2] activity assay (PLAC activity[R], diaDexus) became available, and some laboratories started to measure and report Lp-[PLA.sub.2] activity as an alternative to measurement of concentration (13). The commercial availability of FDA-approved concentration and activity assays presented a dilemma for practitioners because the concordance between the assays was poor. Multiple studies reported correlation coefficients ranging from 0.36-0.86 (7, 11, 12, 14, 15). Although both methods measure the same enzyme, and from first principles should agree well, there has not been a clear explanation for discordance between the 2 assays (12, 16).

LC-MS/MS has been increasingly used for quantifying a variety of analytes in clinical laboratories (17). Such mass spectrometric assays provide improved analytical sensitivity and specificity and have demonstrated value when immunoassays suffer from limited dynamic measuring range or issues with analytical specificity (18) While the analysis of small molecules has been the primary focus of mass spectrometry (MS) in the clinical laboratory, the technique is gaining popularity for the quantification of proteins in plasma or serum (19-22). Measurement of thyroglobulin, a biomarker for thyroid cancer recurrence, is 1 example in which measurement using stable isotope standards and capture by antipeptide antibodies (SISCAPA)-LC-MS/MS immunoaffinity work flow has demonstrated improved accuracy in the presence of antithyroglobulin autoantibodies, a common occurrence that can cause false-negative results in immunoassays (19, 23).

We sought to understand the reason for the discordance between Lp-[PLA.sub.2] activity and concentration and hypothesized that interference in the immunoassay could be responsible. By developing robust MS methods for determination of both enzymatic activity and concentration, we demonstrate that the immunoassays detect only a small and variable fraction of the total Lp-[PLA.sub.2] in a serum sample.

Materials and Methods

HPLC-grade methanol, acetonitrile, and formic acid (FA) were obtained from Fisher. HEPES, sodium chloride, ammonium formate, urea, tris base, iodoacetamide, and tosyl lysyl chloromethyl ketone were purchased from Sigma Aldrich. Lysophosphatidylcholine 17:0 (formula weight 509.7) was purchased from Avanti Polar Lipids. Platelet-activating factor (PAF) C-16-d4 (formula weight 527.7) and lyso PAF C-16 (formula weight 481.7) were purchased from Cayman Chemical. Charcoal-stripped serum was obtained from Golden West Biologicals. A PLAC kit, associated standards, and quality-control pools for measurement of Lp-[PLA.sub.2] concentration were purchased from diaDexus (now Diazyme). An Lp-[PLA.sub.2] Quantikine ELISA kit was purchased from R&D Systems Inc. LipoSepIP[TM], an immunoprecipitation reagent for removal of LDL from human serum, was purchased from Sun Diagnostics. Monoclonal antibody to Lp-[PA.sub.2] tryptic peptide GSVHQNFADFTFATGK was provided by SISCAPA Assay Technologies Ltd. Recombinant native and [sup.15]N-labeled Lp-[PLA.sub.2] was produced by Genscript. Bovine pancreatic trypsin (Cat. No. LS003740) was obtained from Worthington Biochemical Corporation. Remnant clinical samples were used in accordance with Institutional Review Board guidelines at Cleveland HeartLab.

Lp-[PLA.sub.2] PROTEIN QUANTIFICATION BY ELISA

Lp-[PLA.sub.2] concentration in serum specimens was determined by use of a PLAC ELISA kit following the manufacturer's protocol. Lp-[PLA.sub.2] concentrations were calculated from the raw data by use of a calibration curve established with the manufacturer's calibrators. The performance of the assay was monitored by use of the manufacturer's quality-control pools.

To explore PLAC ELISA assay performance in the presence of detergents, minor modifications to the manufacturer's protocol were made. Serum samples were diluted with buffer containing various detergents (to final concentrations of 10 mmol/L CHAPS, 0.25% deoxycholate, 1% Triton X-100 or 0.1% sodium dodecyl sulfate) in 20 mmol/L Tris, pH 8.0. After mixing, samples were incubated at 37 [degrees]C for 1 h before analysis. For the most effective detergent mixture (1%Triton X-100 and 0.25% deoxycholate final concentration), calibrators were diluted similarly to patient samples. To assess the effect of Triton X-100 and deoxycholate detergent mixture on an alternative immunoassay, an R&D systems Lp-[PLA.sub.2] Quantikine ELISA kit was used with similar modifications with a set ofvalidated Lp-[PLA.sub.2] calibrators.

DETERMINATION OF Lp-[PLA.sub.2] ACTIVITY BY MASS SPECTROMETRY

A reaction buffer consisting of 10 mmol/L EDTA, 150 mmol/L NaCl, and 100 mmol/L HEPES (pH 7.2) was used throughout. Deuterated Lp-[PLA.sub.2] substrate, PAF C-16-d4, was prepared at 100 [micro]mol/L in reaction buffer. Lyso PAF C-16 was used as the external calibrator and prepared in charcoal-stripped serum as dilutions in concentrations of 0.5, 1, 2.5, 5, 10, 25, and 50 [micro]mol/L. A stock solution of 50 mmol/L 17:0 lysophosphatidylcholine was prepared in methanol and added into the reaction buffer to a final concentration of 100 [micro]mol/L as the internal standard. Pooled serum samples were measured for their Lp-[PLA.sub.2] activity during the method development and were aliquoted and stored at--70 [degrees]C to be used as quality controls. Serum samples, quality controls, and calibrators were diluted and mixed in the reaction buffer. Diluted serum (10 [micro]L of each) was mixed with 40 [micro]L of Lp-[PLA.sub.2] substrate and incubated at 37 [degrees]C for 10 min. At the end of incubation, the reaction was immediately quenched by the addition of 0.2% FA followed by 50fold dilution with methanol.

LC-MS/MS analysis was performed by an Agilent 1260 Infinity LC system interfaced with an Agilent 6460 Triple Quadrupole mass spectrometer equipped with electrospray ionization. Analytical separation of the Lp[PLA.sub.2] activity product and the internal standard was performed with a normal phase column (Kinetex 2.6 um HILIC 100 A, 50 X 2.1 mm, Phenomenex) and a gradient of 2%-30% solvent A (10% acetonitrile in 100 mmol/L ammonium formate, 0.1% FA) in solvent B (90% acetonitrile, 10% 100 mmol/L ammonium formate, 0.1% FA) over 1 min at 0.6 mL/min. Analytes were quantified via multiple reaction monitoring (MRM) by use of positive-mode electrospray under the following conditions: ionization, 3000 V; sheath gas pressure, 20 psi; sheath gas temperature, 250 [degrees]C. The transitions used were as follows: m/z 486.3 to 184, 510.3 to 184, and 482.3 to 184 for Lp-[PLA.sub.2] product, internal standard, and calibrator, respectively. The analyte peak area to internal standard peak area ratio was plotted against concentration to obtain calibration curves, which were calculated with weighted (1/X) linear, least squares regression. Reduction of raw MS peak areas to concentrations was performed by MassHunter Quantitative Analysis software (Agilent). Results were reported as nmol/min/mL after correction for dilution and reaction time.

Lp-[PLA.sub.2] CONCENTRATION DETERMINED BY SISCAPA IMMUNOAFFINITY MASS SPECTROMETRY

A SISCAPA assay protocol was used to quantify the proteotypic Lp-[PLA.sub.2] tryptic peptide GSVHQNFADFT FATGK in serum sample digests, from which a total protein concentration could be calculated. The protocol made use of a high-affinity mouse monoclonal antipeptide antibody to enrich the Lp-[PLA.sub.2] surrogate peptide from trypsin-digested samples before MS-based quantification. The SISCAPA assays were run with a manual version of the automated work flow previously described but with several modifications (24). Briefly, the antipeptide monoclonal antibody was bound to protein-Gcoated magnetic beads (Life Technologies) to a final concentration of 0.1 [micro]g/mL antibody. A series of calibrators from 100 ng/mL to 2000 ng/mL recombinant Lp-[PLA.sub.2] was prepared in 50 g/L bovine serum albumin by serial dilutions of the high calibrator. Protein concentration of recombinant Lp-[PLA.sub.2] was determined by amino acid analysis before preparation. A total of 30 [micro]L of serum specimens or calibrators were mixed with sufficient reagents to reach final concentrations of 9 mol/L urea, 0.2 mmol/L Tris (pH 8.0), and 0.03 mol/L Tris(2carboxyethyl)phosphine. As an internal standard, 15 [micro]L of 15N-labeled Lp-[PLA.sub.2] (2 [micro]g/mL) were added to each tube. Samples were incubated on a shaker for 30 min before alkylation via 10 min of incubation with iodoacetamide at a final concentration of 25 mmol/L. Sufficient 0.1% CHAPS in 0.2 mol/L Tris (pH 8.0) was added to reduce the urea concentration to 1 mol/L. Trypsin (30 [micro]L; 7 g/L) was added, and samples were digested overnight at 37 [degrees]C. After digestion, freshly prepared 0.6 mol/L tosyl lysyl chloromethyl ketone in 10 mmol/L HCl was added to quench trypsin activity. Then, 10 [micro]L of anti-Lp-[PLA.sub.2] antibody--conjugated magnetic beads were added, and the mixture was placed on a shaker for 2 h. With a magnetic manifold to isolate the beads, the supernatant was discarded and the beads were washed rapidly 3 times with 500 [micro]L of phosphate-buffered saline + 0.03% CHAPS. After the final wash, 50 [micro]L of 0.5% FA + 0.03% CHAPS were added, and the mixture was incubated for 5 min. Tubes were placed on the magnetic holder and the eluent (containing enriched peptides) was transferred to vials for MS assay.

The peptide analytes were separated by reversed phase chromatography with an Agilent 1260 Infinity LC system fitted with a Kinetex LC column (2.6 [micro]m C18 100 [Angstrom], 50 X 3 mm, Phenomenex) and quantified by MRM analysis with an Agilent 6490 Triple Quadrupole mass spectrometer. Analytical separation of the peptides was performed by use of a linear gradient of 1%--35% solvent B (0.1% FA in acetonitrile) in solvent A (0.1% FA in water) over 3.5 min at 0.5 mL/min. The sample injection volume was 20 [micro]L. Analytes were quantified via MRM by positive-mode electrospray under the following conditions: ionization, 3500 V; sheath gas pressure, 25 psi; sheath gas temperature, 250 [degrees]C.

For the tryptic peptide GSVHQNFADFTFATGK, the m/z transitions used were 576.3 [3[H.sup.+]].sup.+3] to 624.3 ([y.sub.6.sup.+]) and 576.3 [[3[H.sup.+]].sup.+3] to 886.4 ([y.sub.8.sup.+]), which were monitored for the quantifier and qualifier ions, respectively. For the internal standard (tryptic peptide from the [sup.15]N-labeled protein), m/z583.3 [[3[H.sup.+]].sup.+3]to 631.3 ([y.sub.6.sup.+]) and 583.3 [[3[H.sup.+]].sup.+3] to 895.4 ([y.sub.8.sup.+]) transitions were used. The analyte peak area to internal standard peak area ratio was plotted against concentration to obtain calibration curves, via weighted (1/x) linear, least squares regression. Reduction of raw MS peak areas to concentrations was performed with MassHunter Quantitative Analysis software.

The limit of quantification (LOQ) was established at the lowest Lp-[PLA.sub.2] concentration, in which the mean imprecision was at 20% CV. A blank sample (50 g/L bovine serum albumin) was supplemented with recombinant Lp-[PLA.sub.2] (20 ng/mL) and diluted linearly to the concentrations of 10, 5, 2.5, 1.25, and 0.25 ng/mL and measured in quadruplicates. Based on interpolation of the measured percent CV, the LOQ was determined as 10 ng/mL. Supplement recovery in patient samples was assessed by adding 300 and 1000 ng/mL of recombinant Lp-[PLA.sub.2], yielding mean recoveries of 95% and 117%, respectively, with a CV of <8.4%. Linearity was assessed by supplementing a serum sample with recombinant Lp[PLA.sub.2] to a final concentration of 1280 ng/mL Lp-[PLA.sub.2] and diluting with stripped serum in 2-fold steps to the LOQ. Comparison of observed to expected Lp-[PLA.sub.2] concentration confirmed linearity across the analytical measurement range with a slope of 1.06 and an [r.sup.2] of 0.99 (see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/ content/vol64/issue4). Two samples with known Lp[PLA.sub.2] concentrations (charcoal-stripped serum supplemented with 300 and 600 ng/mL recombinant Lp[PLA.sub.2]) were used to assess accuracy (see Table 1 in the online Data Supplement for description of accuracy) and precision with 5 replicates over 5 days. Accuracy ranged from 98% to 111% with a mean imprecision of 11% CV.

IMMUNOPRECIPITATION AND WESTERN BLOT ANALYSIS

Serum (100 [micro]L) was mixed with 100 [micro]L 2X detergent mixture (2% Triton X-100, 0.5% sodium deoxycholate, and 20 mmol/L Tris pH 8.0) and incubated at 37 [degrees]C. After 1 h, samples were brought to room temperature and mixed with 100 [micro]L of apolipoprotein B (apoB) precipitant (LipoSepIP[TM], Sun Diagnostics). After a 10-min incubation, samples were centrifuged at 10000g for 10 min to pellet immunoprecipitated low-density lipoprotein, and the supernatant was carefully harvested. The immunoprecipitate was resuspended in 100 [micro]L of sodium dodecyl sulfate (SDS) gel-loading buffer. As controls, serum samples were mixed with phosphatebuffered saline buffer in place of the detergent mixture. A total of 9 [micro]L of each supernatant and 3 [micro]L of the suspended precipitates were denatured, separated on an SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane (Bio-Rad), and probed with an antihuman Lp-[PLA.sub.2] antibody (Cayman Chemical) and antihuman apoB antibody (Abcam) after stripping the membrane with 1.5% Glycine, 0.1% SDS, 1% Tween 20, pH 2.2. HRP-conjugated antirabbit and antigoat antibodies (Abcam) are used as secondary antibodies for Lp-[PLA.sub.2] and apoB blots, respectively. The blots were imaged by chemiluminescence with ECL Western Blotting Substrate (Pierce).

LIPOPROTEIN CHOLESTEROL AND PARTICLE DETERMINATION

Low-density lipoprotein cholesterol (LDL-C) and particle number (LDL-p) and high-density lipoprotein cholesterol (HDL-C) and particle number (HDL-p) content of the samples were determined by nuclear magnetic resonance (NMR Lipoprofile[R], LipoScience, LabCorp).

Results

Lp-[PLA.sub.2] ACTIVITY ASSAY PERFORMANCE

The performance of our laboratory-developed LC-MS/ MS assay for Lp-[PLA.sub.2] activity described in this study was fully assessed and the validation figures ofmerit are found in Table 1 in the online Data Supplement. For method comparison, Lp-[PLA.sub.2] activity determined by this MS assay was compared with the standard FDA-approved colorimetric assay. Our mass MS method correlated to the colorimetric activity assay with an [r.sup.2] = 0.92, n = 70 (see Fig. 2 in the online Data Supplement). Based on comparison to the FDA-approved assay, the clinical cut off value for activity determined by MS was set to 75 nmol/min/mL.

Lp-[PLA.sub.2] ACTIVITY COMPARED TO ELISA-DETERMINED Lp-[PLA.sub.2] CONCENTRATION

Lp-[PLA.sub.2] concentration (measured by the PLAC mass assay) and activity values were determined for 458 patient serum samples. As expected, poor agreement between Lp-[PLA.sub.2] activity and concentration was observed with a Pearson r of 0.39 (Fig. 1) and concomitant discordance in risk categorization. Of the 116 patients in the high-risk category based on the PLAC protein concentration assay cutoffs, 97 had low Lp-[PLA.sub.2] activity. Similarly, 34 of the 53 samples with high Lp-[PLA.sub.2] activity showed low Lp-[PLA.sub.2] concentrations based on the PLAC protein concentration assay. In total, 131 of the 458 (29%) samples were discordant.

Lp-[PLA.sub.2] ACTIVITY COMPARED TO SISCAPA IMMUNO-LC-MS/ MS-DETERMINED Lp-[PLA.sub.2] CONCENTRATION

To assess serum Lp-[PLA.sub.2] concentration by an alternative to the standard immunoassay, a quantitative MS assay using antibody capture of proteotypic surrogate peptides (SISCAPA) was employed (24). Serum specimens that showed a wide range of Lp-[LPA.sub.2] activity (24--114 nmol/mL/min) were selected for this analysis. The Lp-[PLA.sub.2] protein concentration determined by SISCAPA assay showed an agreement with enzyme activity (Pearson r = 0.93, n = 50; Fig. 2). However, protein concentrations measured by immuno-LCMS/MS were much higher than expected based on the observed population range measured with the commercial ELISA assay. As expected, comparison of Lp-[PLA.sub.2] concentrations measured by immuno-LCMS/MS and immunoassay showed a similar discrepancy between the 2 methods (Fig. 3). Consideration of the magnitude of the difference suggested 2 possibilities: improper calibration of the assays or an interference preventing detection of total Lp-[PLA.sub.2] in the immunoassay. In the absence of Lp-[PLA.sub.2] reference material, we decided to analyze the commercial kit calibrator and QC pools using our immune-mass spectrometry approach. Analysis of the PLAC kit calibrators by use of the LC-MS/MS method gave an agreement to the assigned values with a slope of 0.96 and [r.sup.2] of 0.99 (see Fig. 3 in the online Data Supplement), which indicated that kit calibrators had properly assigned protein concentrations. In addition, analysis of the PLAC kit QC pools (142, SE [+ or -] 9; 313, SE [+ or -] 8 ng/mL) agreed with the kit insert and were within the established acceptable range (120--179 and 279--415 ng/mL, respectively).

CORRELATION OF Lp-[PLA.sub.2] WITH LIPOPROTEIN CHOLESTEROL AND PARTICLE CONCENTRATION

Lp-[PLA.sub.2] is associated with different lipoprotein fractions, but the distribution between HDL and LDL for individual patients is unknown (3, 25). Observation that more Lp-[PLA.sub.2] protein was apparently detected by the SISCAPA immunomass spectrometry method than with the commercial ELISA kit prompted us to consider whether interaction of Lp-[PLA.sub.2] with lipoprotein particles could affect epitope availability for the antibodies used in the ELISA. In this case, it is possible that access to the Lp-[PLA.sub.2] epitopes detected by the immunoassay is affected by association with HDL or LDL, causing differential detection. We examined whether the discordance in the ELISA-determined Lp-[PLA.sub.2] concentration and activity could have resulted from such a bias through exploration of the correlations between Lp-[PLA.sub.2] PLAC results, LDL-C, HDL-C, LDL-p, and HDL-p. Association of Lp-[PLA.sub.2] concentration to LDL-C and particle number showed a correlation with a Pearson r of 0.25 and 0.19, respectively (Fig. 4). Similarly, a correlation with a Pearson r of 0.2 was observed between Lp-[PLA.sub.2] concentration and HDL-C; between LP-[PLA.sub.2] and HDL-p, a correlation with Pearson r of 0.07 (Fig. 4). Taken together, these data suggest that PLAC concentration measurement by ELISA does not preferentially detect LDL- or HDL-bound Lp-[PLA.sub.2].

DETERGENTS LIBERATE Lp-[PLA.sub.2] FROM LIPOPROTEINS

Knowing that PLAC Lp-[PLA.sub.2] concentration was essentially independent of lipoprotein concentration led us to consider that liberation of Lp-[PLA.sub.2] from lipoproteins could enhance detection. To investigate this possibility, serum samples were incubated with detergents, and the liberation of Lp-[PLA.sub.2] from lipoproteins was assessed by Western blotting (Fig. 5). Because the majority of Lp-[PLA.sub.2] is associated with LDL (3, 25), a commercially available immunoprecipitation reagent specific for apoB was used for fractionation. In Fig. 5A, the immunoblot shows recombinant and human Lp-[PLA.sub.2] in serum at the expected molecular mass of approximately 50 kDa, with a doublet for native Lp[PLA.sub.2] owing to glycosylation (26). As expected, the supernatants of the apoB-immunoprecipitated samples had no detectable apoB, while apoB was detected in the precipitates, confirming that immunoprecipitation was successful. As a result of association, the presence of Lp-[PLA.sub.2] was, as expected, in the apoB immunoprecipitate of the no-detergent sample and in the supernatant of the detergent-treated sample. Lp-[PLA.sub.2] liberation from apoB-containing lipoproteins was also dependent on detergent concentration (Fig. 5B). Together, these findings indicate that mild detergent treatment fully liberated Lp-[PLA.sub.2] from apoB-containing lipoproteins.

IMPROVED AGREEMENT BETWEEN CONCENTRATION AND ACTIVITY AFTER LIBERATION OF Lp-[PLA.sub.2] FROM LIPOPROTEINS

With the understanding that Lp-[PLA.sub.2] could be liberated from lipoprotein, we tested the effect ofdetergent treatment on the immuno-detection of serum Lp[PLA.sub.2]. Serum Lp-[PLA.sub.2] concentrations in 80 patient samples with and without detergent pretreatment were measured by the PLAC ELISA kit and compared with Lp-[PLA.sub.2] activities measured by SISCAPA immuno mass spectrometry. The presence of detergents at the selected experimental concentrations was demonstrated to have no effect on the standard calibration curve. As expected, the agreement between the concentration and activity was poor (Fig. 6A, Pearson r = 0.48) but dramatically improved upon detergent treatment (Pearson r = 0.97), and total protein concentrations rose substantially to concentrations expected based on earlier evaluation of samples by the LC-MS/MS approach (Fig. 2).

We were interested to determine if the phenomenon of poor detection of the protein analyte by immunoassay was specific to the PLAC assay itself or was general to the immunological detection of Lp-[PLA.sub.2]. By use of an alternative ELISA (R&D Systems) for the detection of Lp-[PLA.sub.2], a similar phenomenon of improved agreement between con centration and activity after detergent treatment was observed (Fig. 6B).

Discussion

Protein quantification in the clinical laboratory has traditionally been performed by immunoassay, with benefits including low cost, high analytical sensitivity, and scalable throughput. However, poor analytical specificity and interferences can compromise the utility of some immunoassays, often in ways that are difficult to detect without a separate, independent comparison assay. The application ofLC-MS/MS as an independent method of protein analysis has revealed such shortcomings in several immunoassays and appears to provide a generally superior alternative for protein quantification (18, 27). For example, LC-MS/MS quantification of thyroglobulin has demonstrated good quantitative performance in the presence of autoantibodies that are well known to substantially compromise immunometric quantification (19). Likewise, in a recent effort to address contradictory data regarding the influence of ethnicity on free Vitamin D, differential response of an immunoassay to various Vitamin D--binding protein isoforms was demonstrated (21). Immunometric detection of Lp-[PLA.sub.2] now joins this growing group of assays, in which protein analysis by LC-MS/MS can reveal and resolve fundamental issues that compromise some immunoassays.

Plasma or serum Lp-[PLA.sub.2] can be determined by concentration or enzymatic activity. While both forms of assay have been commercially available, it is widely recognized that there is poor agreement between the 2, although first principles would dictate agreement between the 2 assuming no effectors, of which none have been reported. For clinicians, this is especially troublesome due to the discordance between a high- and low-risk group assignment based on cutoffs. In this study, 29% of the test population would receive a different risk group assignment depending on the assay used to perform Lp-[PLA.sub.2] measurement.

In contrast to the immunoassay, our SISCAPA-LCMS/MS method to quantify Lp-[PLA.sub.2] at the peptide level demonstrated an agreement with Lp-[PLA.sub.2] activity, while also yielding protein concentration measurements up to 8-fold higher than the corresponding immunoassay measurements. These results indicate that the standard immunoassay does not give accurate quantification of native Lp-[PLA.sub.2] Use of detergent to liberate Lp-[PLA.sub.2] from lipoproteins dramatically enhanced immunodetection and improved the agreement between activity and concentration as determined by immunoassay.

A properly developed LC-MS/MS assay for determination of either Lp-[PLA.sub.2] concentration or activity can provide a robust measurement of Lp-[PLA.sub.2] for use in clinical practice. We suggest that Lp-[PLA.sub.2] provides an object lesson in the importance of using multiple independent analytical tools to thoroughly test assumptions commonly made during assay development.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: C. Topbas, Cleveland Heart Lab; A. Swick, Cleveland Heartlab; M. Razavi, SISCAPA Assay Technologies;

N.L. Anderson, SISCAPA Assay Technologies, Inc.; T.W. Pearson, SISCAPA Assay Technologies, Inc.; C. Bystrom, Cleveland HeartLab. Consultant or Advisory Role: T.W. Pearson, SISCAPA Assay Technologies, Inc.

Stock Ownership: N.L. Anderson, SISCAPA Assay Technologies, Inc.; T.W. Pearson, SISCAPA Assay Technologies, Inc.; C. Bystrom, Cleveland HeartLab.

Honoraria: None declared.

Research Funding: C. Bystrom, research funded by employer.

Expert Testimony: None declared.

Patents: C. Topbas, CHL-35104/Us-1/Pro; N.L. Anderson, 7,632,686; C. Bystrom, 62/449,898.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or final approval of manuscript.

Acknowledgments: The authors thank Cleveland HeartLab personnel for their technical assistance and critical review. The authors also thank Richard Yip and Matt Pope at SISCAPA Assay Technologies Inc. for technical and organizational assistance.

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Celalettin Topbas, [1] Alan Swick, [1] Morteza Razavi, [2] N. Leigh Anderson, [2] Terry W. Pearson, [2] and Cory Bystrom [1] *

[1] Cleveland HeartLab, Cleveland, OH; [2] SISCAPA Assay Technologies, Inc., Washington, DC.

* Address correspondence to this author at: 6701 Carnegie, Suite 500, Cleveland, OH 44103. Fax +866-449-0960; e-mail cbystrom@clevelandheartlab.com.

Received July 27,2017; accepted December 18,2017.

Previously published online at DOI: 10.1373/clinchem.2017.279752

[3] Nonstandard abbreviations: Lp-[PLA.sub.2], lipoprotein-associated phospholipase 2; FDA, Food and Drug Administration; MS, mass spectrometry; SISCAPA, stable isotope standards and capture by anti-peptide antibody; FA, formic acid; PAF, Platelet-activating factor; MRM, multiple reaction monitoring; LOQ, limit of quantification; SDS, sodium dodecyl sulfate; apoB, apolipoprotein B; LDL-C, low-density lipoprotein cholesterol; LDL-p, low-density lipoprotein particle number; HDL-C, high-density lipoprotein cholesterol; HDL-p, high-density lipoprotein particle number.

Caption: Fig.1. Correlation of the Lp-[PLA.sub.2] activity measured by mass spectrometry vs concentration measured by PLAC mass assay.

Shaded areas show the discordant test results.

Caption: Fig. 2. Correlation of Lp-[PLA.sub.2] activity vs concentration measured by mass spectrometry.

Caption: Fig. 3. Correlation of the Lp-[PLA.sub.2] concentration measured By SISCAPA assay (immunomass spectrometry) vs PLAC assay (immunoassay).

Caption: Fig. 4. Relationship between the Lp-[PLA.sub.2] concentration (measured by PLAC mass assay) and different lipoprotein fractions.

Caption: Fig.5. Western blot analysis of detergent-treated samples.

Serum samples with and without detergent treatment were immunoprecipitated with LipoSep (Sun Diagnostics); fractions were run on gradient-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed for apoB and Lp-[PLA.sub.2]. Top panel, Western blot analysis with anti-apoB antibody; bottom panel, with anti-Lp-[PLA.sub.2] antibody (A). M, molecular weight marker; 1, recombinant Lp-[PLA.sub.2]; 2, serum; 3, untreated serum supernatant; 4, untreated serum immunoprecipitate; 5, treated serum supernatant; 6, treated serum immunoprecipitate. Immunoprecipitated serum supernatants containing increasing detergent concentrations were probed with anti-Lp-[PLA.sub.2] antibody(B).

Caption: Fig.6. Correlation of Lp-[PLA.sub.2] activity vs concentration.

Lp-[PLA.sub.2] activity vs concentration, measured by PLAC mass assay (A). Solid boxes indicate results obtained by the original assay and open circles indicate results obtained after detergent treatment. Activity vs concentration, measured by an alternative ELISA kit (R&D systems) (B). Solid boxes show results obtained with samples incubated in phosphate-buffered saline and open circles show results obtained after detergent treatment.
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Title Annotation:Proteomics and Protein Markers
Author:Topbas, Celalettin; Swick, Alan; Razavi, Morteza; Anderson, N. Leigh; Pearson, Terry W.; Bystrom, Co
Publication:Clinical Chemistry
Date:Apr 1, 2018
Words:5549
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