Abstract
Microparticles circulate in plasma and have recently emerged as potential inflammatory markers in cardiovascular disease. They are fragments of cell membranes that express cluster of differentiation (CD) antigens and are present at elevated levels in patients with acute coronary syndrome. We have developed a novel method for the rapid detection of microparticles in plasma using a fluorescence-based antibody array system. Isolated microparticles are captured on anti-CD antibody spots immobilized on a nitrocellulose membrane. These CD antibodies are directed against extracellular epitopes, whereas the intracellular exposed surface of the microparticles is labeled with a fluorescent anti-annexin antibody. The array is then scanned and quantified. A pilot study was undertaken to compare microparticle CD antigen expression in acute coronary syndrome and healthy subjects. Ten CD antigens (44, 45, 54, 62E, 79, 102, 117, 130, 138, and 154) had significantly increased expression in the disease group relative to the healthy controls. These results were then verified using flow cytometry and scanning electron microscopy. Although we have focused our analysis on changes in microparticle CD antigen expression, this technique is amenable to analyzing other surface markers. Microparticles can be derived from a wide variety of cell types, so selection of the primary antibody can be tailored to the cell origin that is to be investigated.
Highlights
Microparticles circulate in plasma and have recently emerged as potential inflammatory markers in cardiovascular disease
Test antibody arrays were constructed consisting of CD44 and CD45 antibodies to determine whether microparticles isolated from plasma can be captured and imaged
Construction of the Test Antibody Array—Each test array consisted of a virgin nitrocellulose slide onto which were placed duplicate anti-CD44 spots and a single anti-CD45
Summary
This process of adherence and migration is dependent on a range of cell membrane receptors known as CD antigens. Because microparticles are fragments of cell membranes they express CD antigens, and elevated levels are present in patients with coronary artery disease compared with age-matched healthy controls (1). Using the proteomic expression on an external cell membrane, the origin of the microparticles can be determined by using specific antibodies against epitopes located on these membranes. These can be detected using flow cytometry or ELISA. We found increased expression levels of several different CD antigens in the disease group compared with samples from healthy subjects These results were verified using flow cytometry and electron microscopy
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