ELISPOT
Encyclopedia
The Enzyme-linked immunosorbent spot (ELISPOT) assay is a common method for monitoring immune responses in humans and animals. It was developed by Cecil Czerkinsky in 1983.
The ELISPOT assay is based on, and was developed from a modified version of the ELISA
immunoassay
. ELISPOT assays were originally developed to enumerate B cell
s secreting antigen-specific antibodies
, and have subsequently been adapted for various tasks, especially the identification and enumeration of cytokine
-producing cells at the single cell level. Simply put, at appropriate conditions the ELISPOT assay allows visualization of the secretory product of individual activated or responding cells. Each spot that develops in the assay represents a single reactive cell. Thus, the ELISPOT assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information.
By virtue of exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific responses) which were not possible before are now relatively easy. This exceptional sensitivity is in part because the product is rapidly captured around the secreting cell: before it is either diluted in the supernatant, captured by receptors of adjacent cells, or degraded. This makes ELISPOT assays much more sensitive than conventional ELISA measurements. Limits of detection are below 1/100,000 rendering enumerate the actively producing cells. This allows much of the analysis process to be automated, and permits a greater level of accuracy than what can be achieved using manual inspection.
) technique. Either a monoclonal
(preferred for greater specificity) or polyclonal capture antibody that is coated aseptically onto a PVDF (polyvinylidene fluoride
) -backed microplate. These antibodies are chosen for their specificity for the analyte in question.
The plate is blocked, usually with a serum protein that is non-reactive with any of the antibodies in the assay. After this, cells of interest are plated out at varying densities, along with antigen
or mitogen
, and then placed in a humidified 37°C CO2
incubator for a specified period of time.
Cytokine (or other cell product of interest) secreted by activated cells is captured locally by the coated antibody on the high surface area PVDF membrane. After washing the wells to remove cells, debris, and media components, a biotin
ylated polyclonal antibody specific for the chosen analyte is added to the wells. This antibody is reactive with a distinct epitope of the target cytokine and thus is employed to detect the captured cytokine. Following a wash to remove any unbound biotinylated antibody, the detected cytokine is then visualized using an avidin
-HRP
, and a precipitating substrate (e.g., AEC, BCIP
/NBT
). The colored end product (a spot, usually a blackish blue) typically represents an individual cytokine-producing cell. The spots can be counted manually (e.g., with a dissecting microscope) or using an automated reader to capture the microwell images and to analyze spot number and size.
assay
is a modification of the ELISPOT assay and is based on using multiple fluorescent anticytokines which makes it possible to spot two cytokines in the same assay.
The ELISPOT assay is based on, and was developed from a modified version of the ELISA
ELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
immunoassay
Immunoassay
An immunoassay is a biochemical test that measures the presence or concentration of a substance in solutions that frequently contain a complex mixture of substances. Analytes in biological liquids such as serum or urine are frequently assayed using immunoassay methods...
. ELISPOT assays were originally developed to enumerate B cell
B cell
B cells are lymphocytes that play a large role in the humoral immune response . The principal functions of B cells are to make antibodies against antigens, perform the role of antigen-presenting cells and eventually develop into memory B cells after activation by antigen interaction...
s secreting antigen-specific antibodies
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
, and have subsequently been adapted for various tasks, especially the identification and enumeration of cytokine
Cytokine
Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...
-producing cells at the single cell level. Simply put, at appropriate conditions the ELISPOT assay allows visualization of the secretory product of individual activated or responding cells. Each spot that develops in the assay represents a single reactive cell. Thus, the ELISPOT assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information.
By virtue of exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific responses) which were not possible before are now relatively easy. This exceptional sensitivity is in part because the product is rapidly captured around the secreting cell: before it is either diluted in the supernatant, captured by receptors of adjacent cells, or degraded. This makes ELISPOT assays much more sensitive than conventional ELISA measurements. Limits of detection are below 1/100,000 rendering enumerate the actively producing cells. This allows much of the analysis process to be automated, and permits a greater level of accuracy than what can be achieved using manual inspection.
Procedure
As noted above, the ELISPOT assays employ a technique very similar to the sandwich enzyme-linked immunosorbent assay (ELISAELISA
Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
) technique. Either a monoclonal
Monoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
(preferred for greater specificity) or polyclonal capture antibody that is coated aseptically onto a PVDF (polyvinylidene fluoride
Polyvinylidene fluoride
Polyvinylidene fluoride, or PVDF is a highly non-reactive and pure thermoplastic fluoropolymer.PVDF is a specialty plastic material in the fluoropolymer family; it is used generally in applications requiring the highest purity, strength, and resistance to solvents, acids, bases and heat and low...
) -backed microplate. These antibodies are chosen for their specificity for the analyte in question.
The plate is blocked, usually with a serum protein that is non-reactive with any of the antibodies in the assay. After this, cells of interest are plated out at varying densities, along with antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
or mitogen
Mitogen
A mitogen is a chemical substance that encourages a cell to commence cell division, triggering mitosis. A mitogen is usually some form of a protein.Mitogenesis is the induction of mitosis, typically via a mitogen....
, and then placed in a humidified 37°C CO2
Carbon dioxide
Carbon dioxide is a naturally occurring chemical compound composed of two oxygen atoms covalently bonded to a single carbon atom...
incubator for a specified period of time.
Cytokine (or other cell product of interest) secreted by activated cells is captured locally by the coated antibody on the high surface area PVDF membrane. After washing the wells to remove cells, debris, and media components, a biotin
Biotin
Biotin, also known as Vitamin H or Coenzyme R, is a water-soluble B-complex vitamin discovered by Bateman in 1916. It is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring...
ylated polyclonal antibody specific for the chosen analyte is added to the wells. This antibody is reactive with a distinct epitope of the target cytokine and thus is employed to detect the captured cytokine. Following a wash to remove any unbound biotinylated antibody, the detected cytokine is then visualized using an avidin
Avidin
Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds, reptiles and amphibians deposited in the whites of their eggs. In chicken egg white, avidin makes up approximately 0.05% of total protein...
-HRP
Horseradish peroxidase
The enzyme horseradish peroxidase , found in horseradish, is used extensively in biochemistry applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule.-Applications:...
, and a precipitating substrate (e.g., AEC, BCIP
5-Bromo-4-chloro-3-indolyl phosphate
5-Bromo-4-chloro-3-indolyl phosphate is a chemical compound used in immunoblotting, in situ hybridization, and immunohistochemistry, with nitro blue tetrazolium chloride , for sensitive colorimetric detection of alkaline phosphatase. BCIP is the alkaline phosphatase substrate, which is...
/NBT
Nitro blue tetrazolium chloride
Nitro blue tetrazolium is a chemical compound composed of two tetrazole moieties. It is used in immunology for sensitive detection of alkaline phosphatase . NBT serves as the oxidant and BCIP is the AP-substrate ....
). The colored end product (a spot, usually a blackish blue) typically represents an individual cytokine-producing cell. The spots can be counted manually (e.g., with a dissecting microscope) or using an automated reader to capture the microwell images and to analyze spot number and size.
Fluorospot Assay
The FluoroSpotFluoroSpot
The FluoroSpot assay is a modification of the ELISPOT assay. ELISPOT usually measures the cellular secretion of one cytokine in each well by the use of an enzyme-labeled anti-cytokine antibody. FluoroSpot measures the secretion of several cytokines in each well by the use of fluorescent...
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...
is a modification of the ELISPOT assay and is based on using multiple fluorescent anticytokines which makes it possible to spot two cytokines in the same assay.