Posts Tagged ‘analysis’

Question by James: Apart from ELISA, which other immunoassay(s) can be used for quantitative antigen analysis?
to investigate immunological disease…….

Best answer:

Answer by M A S
To investigate whether immunologic abnormalities in patients with Behçet’s disease (BD) are related to abnormalities of the Th1/Th2 ratio.

Recrudescence and Its Use to Investigate the Role of Immune Cells in Prevention of Recurrent Disease ANTHONY SIMMONS* AND ANTHONY A.

The role of adrenocorticoids as modulators of immune function
in health and disease: neural, endocrine and immune interactions.

Detecting the quantity of antibody or antigen can be achieved by a variety of methods. ELISA is a commonly used technique for detecting antibody or antigen quantity. One of the most common methods is to label either the antigen or antibody with either an enzyme (see enzyme immunoassay, EIA), radioisotopes such as I-125 radioimmunoassay (RIA) or fluorescence. Other techniques include agglutination, nephelometry, turbidimetry, Western blot, immunoprecipitation, immunocytochemistry, immunohistochemistry, ELISPOT, flow cytometry, Luminex assay, Cytometric Bead Array, and others.

through bioconjugation.

Radioimmunoassay – (RIA) involves mixing known quantities of radioactive antigen (frequently labeled with gamma-radioactive isotopes of iodine attached to tyrosine) with antibody to that antigen, then adding unlabeled or “cold” antigen and measuring the amount of labeled antigen displaced. Initially, the radioactive antigen is bound to the antibodies. When “cold” (unlabeled, quest) antigen is added, the two compete for antibody binding sites – at higher concentrations of “cold” antigen, more of it binds to the antibody, displacing the radioactive variant. The bound antigens are separated from the unbound ones.

ELISpot – (Enzyme-Linked Immunosorbent Spot assay) is based on, and was developed from a modified version of the ELISA immunoassay. ELISpot assays were originally developed to enumerate B cells 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.

Immunoprecipitation – (IP) is the technique of precipitating an antigen out of solution using an antibody specific to that antigen. This process can be used to enrich a given protein to some degree of purity. Co-immunoprecipitation (also known as a ‘pull-down’) can identify protein complexes present in cell extracts: by IPing one protein believed to be in a complex, additional members of the complex can also be identified. The complexes are brought out of solution by insoluble antibody-binding proteins isolated initially from bacteria, such as Protein A or Protein G. These can also be coupled to agarose beads that can easily be isolated out of solution. After washing, the precipitate can be analyzed using gel electrophoresis, mass spectrometry, western blotting, or any number of other methods for identifying constituents in the complex. Thus, co-immunoprecipitation is a standard method to assess protein-protein interaction.

Immunocytochemistry – (ICC) includes using antibodies to detect antigen in cell (on the cell surface or within the cell). The antigen is stained with a primary antibody, which bonds directly to the antigen, followed by a secondary antibody, which bonds to the primary antibody. Next, a tertiary reagent is applied, which bonds to the secondary antibody, with an enzymatic end. When the quarternary reagent, or substrate, is applied, the enzymatic end of the tertiary reagent converts the substrate into a pigment and another product, which stains the cell, usually a reddish brown color.

Immunohistochemistry – (IHC) refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues. It takes its name from the roots “immuno,” in reference to antibodies used in the procedure, and “histo,” meaning tissue. Immunohistochemical staining is widely used in the diagnosis and treatment of cancer. Specific molecular markers are characteristic of particular cancer types. IHC is also widely used in basic research to understand the distribution and localization of biomarkers in different parts of a tissue. Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyze a color-producing reaction (see immunoperoxidase staining). Alternatively, the antibody can also be tagged to a fluorophore, such as FITC, rhodamine, or Texas Red, (see immunofluorescence). The latter method is of great use in confocal laser scanning microsco

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