Celalettin Topbas (Presenter)
Cleveland Heart Lab
Bio: Celalettin Topbas is a postdoctoral research scientist at Cleveland Heart Lab. He received his B.S degree in Molecular Biology from the Bogazici University, Istanbul and Ph.D. in Clinical Bioanalytical Chemistry from Cleveland State University. His academic experience and interest includes analytical chemistry, mass spectrometry and protein chemistry.
Authorship: Celalettin Topbas(1), Morty Razavi(2), Leigh Anderson(2),Cory Bystrom (1)
(1) Cleveland Heart Lab, (2) SISCAPA Assay Technologies
| Short Abstract Lipoprotein Associated Phospholipase A2 (Lp-PLA2), is an enzyme associated with vascular inflammation. Both the concentration and the activity of Lp-PLA2 in serum are used clinically to assess risk of coronary heart disease. We compared an in-house proteomic LC-MS/MS (SISCAPA®) concentration assay, a fully validated LDT for LP-PLA2 activity and the FDA approved Lp-PLA2 concentration assays to investigate their relationship. The ELISA showed a weak correlation with both concentration and activity while the first two compared well. Further investigation revealed that the immunoassay suffers from a substantial interference and unpredictably detects only a fraction of the total Lp-PLA2 in human serum. |
Long Abstract
Background: Lipoprotein Associated Phospholipase A2 (Lp-PLA2), an enzyme involved in metabolism of lipids and inflammation, is a novel biomarker for Cardiovascular Disease. Both the concentration and the activity of Lp-PLA2 have been shown to be associated with risk of coronary heart disease. While enzyme linked immunoassays are utilized to quantitate plasma Lp-PLA2 concentration, radiometric and colorimetric assays have been used to measure the activity of this enzyme. We have developed two LC/MS based assays to quantitate the activity and the mass of Lp-PLA2 in plasma and investigated their correlation to a commonly used FDA approved immunoassay (PLAC® by Diadexus, Inc.).
Methods: PLAC assay: The Lp-PLA2 mass (concentration) in samples were quantified by using the PLAC test by following the manufacturers protocol. The test uses two monoclonal anti-Lp-PLA2 antibodies in a sandwich immunoassay format combined with HRP- tetramethylbenzidine detection system. Lp-PLA2 concentrations were determined by using a six-point standard curve using manufacturer reagents.
Lp-pLA2 activity assay: We have developed and validated a novel LC/MS method to measure Lp-PLA2 activity in plasma. Briefly, samples were incubated with Lp-PLA2 substrate, the reaction was quenched and the product was extracted into methanol. A seven-point calibration curve was constructed and used to quantitatively determine the amount of product. The analytes were separated by a normal phase chromatography (Kinetex® HILIC column) and quantified using a multiple reaction monitoring (MRM). The LDT assay was validated according to CLIA/CAP guidelines.
Lp-PLA2 mass assay: An optimized Lp-PLA2 concentration assay utilizing an anti-peptide antibody enrichment (SISCAPA®) workflow method was developed. Briefly, samples were spiked with known amounts of N15 labelled recombinant Lp-PLA2 and denatured and digested with trypsin. Upon quenching the protease, the peptide mix was incubated with anti-Lp-PLA2 peptide antibody (SISCAPA®) coupled with Protein G coated magnetic beads (Life Technologies). A six-point calibration curve was established using known amounts of recombinant Lp-PLA2.
Results: We observed a strong correlation (r=0.96) between the LC-MS/MS Lp-PLA2 activity assay and the established colorimetric activity assay. Likewise, Lp-PLA2 activity correlates well with the Lp-PLA2 concentration when determined by LC-MS/MS method (r=0.97). However, the immunoassay fails to quantify total Lp-PLA2 and shows a very weak correlation to mass determined by proteomic techniques and activity determined by LC-MS/MS. This strongly suggests that the commercial ELISA detects an immunoreactive species that is substantially unrelated to protein abundance.
Conclusion: The well documented discordance between the Lp-PLA2 concentration and activity assays appears to be related to a fundamental disconnection between immunoreactivity and abundance. The exact nature of the interference in the concentration assay is unknown but it suggests the existence of a protein-complex, immune-complex or conformational state that obscures the epitope. However, activity is closely related to the true concentration of the protein in serum as measured using proteomic techniques. Properly employed quantitative analysis of LP-PLA2 by LC-MS/MS provided key insight in determining the true concentration of LP-PLA2 in serum, demonstrating the value of proteomic workflows in assessment and development of clinical markers.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Cleveland Heart Lab |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |