Annexin A5 affinity assay
Encyclopedia
In molecular biology, an annexin A5 affinity assay is a test to quantify the number of cells undergoing apoptosis
. The assay
uses the protein annexin A5
to tag apoptotic and dead cells and the numbers are then counted using either flow cytometry
or a fluorescence microscope
.
The annexin a5 protein binds to apoptotic cells in a calcium dependent manner using phosphatidylserine containing membrane surfaces which are usually only present on the inner leaflet of the membrane.
is a form of programmed cell death which is used by the body to remove unwanted, damaged or senescent cells from tissues. Removal of apoptotic cells is carried out via phagocytosis by white blood cells such as macrophages or dendritic cells. Phagocytic white blood cells recognize apoptotic cells by their exposure of negatively charged phospholipids(phosphatidylserine) on the cell surface.
In normal cells the negative phospholipids reside on the inner side of the cellular membrane and the outer surface of the membrane is occupied by phospholipids which do not have a charge. After a cell has entered apoptosis, the negatively charged phospholipids are transported to the outer cell surface by a hypothetical protein known as scramblase
. Phagocytic white blood cells express a receptor which can detect the negatively charged phospholipids. After detection the apoptotic cells are removed.
if it occurs on cells which are in the early phase of executing cell death. Internalization amplifies additionally the intensity of the annexin A5 stained cell.
Annexin A5 has been successively used to detect apoptotic cells in vitro and in vivo. Pathological processes in which apoptosis occurs include inflammation, ischemia damage of the heart caused by myocardial infarction, apoptotic white blood cells and smooth muscle cells present in atherosclerotic plaques of blood vessels, transplanted organs in the donor patient which are rejected by the immune system or tumour cells which are exposed to cytostatic drugs during chemotherapy.
The non-invasive detection of diseased tissue with, for example, radioactively labeled annexin A5 is the goal of a recently developed line of research known as Molecular Imaging.
Molecular Imaging of cell death using radioactive annexin A5 can become of clinical significance to diagnose vulnerability of atherosclerotic plaques (unstable atherosclerosis
), heart failure, transplant rejection
, and to monitor efficacy of anti-cancer
therapy.
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
. The assay
Assay
An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...
uses the protein annexin A5
Annexin A5
Annexin A5 is a cellular protein in the annexin group. The function of the protein is unknown; however, annexin A5 has been proposed to play a role in the inhibition of blood coagulation by competing for phosphatidylserine binding sites with prothrombin and also to inhibit the activity of...
to tag apoptotic and dead cells and the numbers are then counted using either flow cytometry
Flow cytometry
Flow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...
or a fluorescence microscope
Fluorescence microscope
A fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption...
.
The annexin a5 protein binds to apoptotic cells in a calcium dependent manner using phosphatidylserine containing membrane surfaces which are usually only present on the inner leaflet of the membrane.
Background
ApoptosisApoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
is a form of programmed cell death which is used by the body to remove unwanted, damaged or senescent cells from tissues. Removal of apoptotic cells is carried out via phagocytosis by white blood cells such as macrophages or dendritic cells. Phagocytic white blood cells recognize apoptotic cells by their exposure of negatively charged phospholipids(phosphatidylserine) on the cell surface.
In normal cells the negative phospholipids reside on the inner side of the cellular membrane and the outer surface of the membrane is occupied by phospholipids which do not have a charge. After a cell has entered apoptosis, the negatively charged phospholipids are transported to the outer cell surface by a hypothetical protein known as scramblase
Scramblase
Scramblase is a protein responsible for the translocation of phospholipids between the two monolayers of a lipid bilayer of a cell membrane. In humans, phospholipid scramblases constitute a family of five homologous proteins that are named as hPLSCR1–hPLSCR5. Scramblases are members of the...
. Phagocytic white blood cells express a receptor which can detect the negatively charged phospholipids. After detection the apoptotic cells are removed.
Detection of cell death with annexin A5
Healthy individual apoptotic cells are rapidly removed by phagocytes. However in pathological processes the removal of apoptotic cells may be delayed or even absent. Dying cells in tissue can be detected with annexin A5. Labeling of annexin A5 with fluorescent or radioactive molecules makes it possible to detect binding of labeled annexin A5 to the cell surface of apoptotic cells. After binding to the phospholipid surface, annexin A5 assembles into a trimeric cluster. This trimer consists of three annexin A5 molecules which are bound to each other via non-covalent protein-protein interactions. The formation of annexin A5 trimers results in the formation of a two-dimensional crystal lattice on the phospholipid membrane. This clustering of annexin A5 on the membrane greatly increases the intensity of annexin A5 when labeled with a fluorescent or radioactive probe. Two-dimensional crystal formation is believed to cause internalization of annexin A5 through a novel process of endocytosisEndocytosis
Endocytosis is a process by which cells absorb molecules by engulfing them. It is used by all cells of the body because most substances important to them are large polar molecules that cannot pass through the hydrophobic plasma or cell membrane...
if it occurs on cells which are in the early phase of executing cell death. Internalization amplifies additionally the intensity of the annexin A5 stained cell.
Annexin A5 has been successively used to detect apoptotic cells in vitro and in vivo. Pathological processes in which apoptosis occurs include inflammation, ischemia damage of the heart caused by myocardial infarction, apoptotic white blood cells and smooth muscle cells present in atherosclerotic plaques of blood vessels, transplanted organs in the donor patient which are rejected by the immune system or tumour cells which are exposed to cytostatic drugs during chemotherapy.
The non-invasive detection of diseased tissue with, for example, radioactively labeled annexin A5 is the goal of a recently developed line of research known as Molecular Imaging.
Molecular Imaging of cell death using radioactive annexin A5 can become of clinical significance to diagnose vulnerability of atherosclerotic plaques (unstable atherosclerosis
Atherosclerosis
Atherosclerosis is a condition in which an artery wall thickens as a result of the accumulation of fatty materials such as cholesterol...
), heart failure, transplant rejection
Transplant rejection
Transplant rejection occurs when transplanted tissue is rejected by the recipient's immune system, which destroys the transplanted tissue. Transplant rejection can be lessened by determining the molecular similitude between donor and recipient and by use of immunosuppressant drugs after...
, and to monitor efficacy of anti-cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
therapy.