Date of Award

Spring 5-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Cardiovascular disease (CVD) is the leading cause of morbidity, mortality, and health care costs in the developed world and is often undiagnosed until clinical presentation with a major adverse cardiovascular event (MACE). Plasma myeloperoxidase (MPO) content is an emerging biomarker for risk, progression, and prognosis at different stages of CVD. Enzyme-linked immunosorbent assays (ELISAs) are currently used to measure clinical plasma MPO concentration, but ELISAs are costly and time-intensive. Luminol is a chemiluminescent compound with specificity for MPO activity in vivo, but is not sensitive enough for use as a bioluminescence reporter of plasma MPO oxidation. The luminol derivative L-012 is more sensitive to oxidation, but little is known about its sensitivity or specificity to reactive oxygen species (ROS) produced by MPO and its interactions with the myriad components of whole plasma. Therefore L-012 was first characterized as an MPO-dependent bioluminescence reporter in whole blood and plasma. The data indicated that L-012 is not a reporter of all MPO-dependent reactions, but specifically reports halogenation with chloride or bromide. Additionally, plasma components inhibited MPO both specifically and non-specifically and prevented precision measurement of plasma MPO activity with L-012 bioluminescence. To overcome this, a method was developed to isolate MPO on a solid substrate, remove small molecule antioxidants, and eliminate specific inhibition by plasma proteins. With this method, MPO activity from human samples could be assayed using bovine plasma supplemented with purified human MPO as calibration standards. The assay can be performed in under an hour, comprises only two steps, and uses no costly immunologic reagents. The assay was validated with a pilot study using 72 plasma samples from cardiology patients undergoing elective catheterization. Individuals in this cohort assayed by the bioluminescence method were concordant with a parallel ELISA within 2 ± 11 μg/L MPO and overall the measurements from the two assays were not significantly different. To further validate the assay, an outside laboratory also measured MPO from 67 of the same plasma samples with an approved, clinical MPO ELISA. The newly developed MPO assay agreed with the outside ELISA on par with the parallel ELISA. In conclusion, a clinical assay for plasma MPO activity was developed with comparable sensitivity and specificity to current ELISAs which can be performed at a fraction of the time and cost.

Language

English (en)

Chair and Committee

David Piwnica-Worms

Committee Members

Samuel Achilefu, Mary Dinaur, Sergej Djuranovic, Daniel Ory

Comments

Permanent URL: https://doi.org/10.7936/K74J0C84

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