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Knowledge in Immunity
RADIOIMMUNOESSAY
Besides fluorescent dyes, many other distinctive Labels also can be conjugated to antigens and antibodies.The most commonly used labels are radioisotopes and enzymes. A variety of tests have been devised for themeasurement of antigens and antibodies using such labeled reactant. The term binder-ligand-assay has been used for these reactions. The substance (antigen) whose concentration is to be determined is termed the analyte or ligand. The binding protein (ordinarily, the antibody) which binds to the land is called the binderThe first reaction of this type was radioimmunoassay (RIA) described by Benson and Yalow in 1939 RIA (108 ) quantities. RIA and its modifications have permits the measurement of analytes up to picogramversatile applications in various areas of biology and medicine, including the quantitation of hormones drugs, tumour markers, IgE and viral ang The importance of RIA was acknowledged when the Nobel Prize was awarded to Yallow for its discoveryin 1977.RIA is a competitive binding assay in which fined amounts of antibody and radiolabeled antigen in the presence of unlabeled antigen. The labeled and unlabeled antigens compete for the limited binding sites on the antibody. This competition is determined by the level of the unlabeled test) antigen presentthe reacting system. After the reaction, the antigen separated into free and bound fractions and the radioactive counts measured. The concentration the test antigen can be calculated from the ratio of thebound and total antigen labels, using a standard dose response curveFor any reacting system, the standard dose response or calibration curve has to be prepared first. This is done by running the reaction with fixed amount of antibodyand labeled antigen, and carving known amounts of unlabeled antigen. The ratio of bound: total labels (8:T ratio) plotted against the analyte concentrations give the standard calibration curve. The concentration of antigen in the test sample is computed from theBT ratio of the test by interpolation from thecalibration curve.
Yamini Chandru
IMMUNOPRECIPITATION
The immunoprecipitation technique has the advantage of allowing the isolation of the antigen of interest for further analysis. It also provides a sensitive assay for the presence of aparticular antigen in a given cell or tissue type. An extract produced by disruption of cells or tissues is mixed with an antibody against the antigen of interest in order to form an antigen-antibody complex that will precipitate. However, if the antigen concentration is low (often the case in cell and tissue extracts), the assembly of antigen-antibody complexes into precipitates can take hours, even days, and it is difficult to isolate the small amount of immuno precipitate that forms. Fortunately, there are a number of ways to avoid these limitations. One is to attach the antibody to a solid support,such as a synthetic bead, which allows the antigen-antibody complex to be collected by centrifugation. Another is to adda secondary antibody specific for the primary antibody to bind the antigen-antibody complexes. If the secondary antibody is attached to a bead, the immune complexes can be collected by centrifugation. A particularly ingenious version of this procedure involves the coupling of the secondary antibody to magnetic beads. After the secondary antibody binds to the primary antibody, immunoprecipitates are collected by placing a magnet against the side of the tubeWhen used in conjunction with biosynthetic radioisotope labeling, immunoprecipitation can also be used to determine whether a particular antigen is actually synthesized by a caltor tissue. Radiolabeling of proteins synthesized by cells of interest can be done by growing the cells in cell-culture medium containing one or more radiolabeled amino acids.Generally, the amino acids used for this application are those most resistant to metabolic modification, such as leucine.cysteine, or methionine. After growth in the radioactive medium, the cells are lysed and subjected to a primary antibody specific for the antigen of interest. The Ag-Ab complexis collected by immunoprecipitation, washed free of union corporated radiolabeled amino acid and other impurities, and then analyzed. The complex can be counted in a scintillation counter to obtain a quantitative determination of the amount of the protein synthesized. Further analysis often involves disruption of the complex, usually by use of SDS and heat, so that the identity of the immunoprecipitated antigencan be confirmed by checking that its molecular weight is that expected for the antigen of interest. This is done by separation of the disrupted complex by SDS-PAGE and subsequent autoradiography to determine the position of the radiolabeled antigen on the gel.