Using Antibody Arrays to Detect Microparticles from Acute Coronary Syndrome Patients Based on Cluster of Differentiation (CD) Antigen Expression

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. 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. Microparticles are membrane fragments derived mainly from platelets and endothelial cells but also from leukocytes, smooth muscle cells, and erythrocytes. They are formed during apoptosis and range in size from 0.1 to 1.0 μm. Their outer leaflets contain phosphatidylserine, which may be labeled with the phospholipid probe annexin V. Microparticles circulate in plasma and have recently emerged as potential inflammatory markers in cardiovascular disease (1Boulanger C.M. Amabile N. Tedgui A. Circulating microparticles: a potential prognostic marker for atherosclerotic vascular disease.Hypertension. 2006; 48: 180-186Crossref PubMed Scopus (331) Google Scholar). Coronary artery disease is caused by atherosclerosis, a disease of the large arteries (2Lusis A.J. Atherosclerosis.Nature. 2000; 407: 233-241Crossref PubMed Scopus (4735) Google Scholar). It can manifest as stable angina or as acute coronary syndrome (ACS). 1The abbreviations used are: ACS, acute coronary syndrome; CD, cluster of differentiation; NO, nitric oxide; PDP, platelet-depleted plasma. The latter is a broad term describing a group