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Transcript
Radioimmunoassay • Radioimmunoassay (RIA) involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantitation using radioactivity. • The technique of radioimmunoassay has revolutionized research and clinical practice in many areas, e.g., – blood banking – diagnosis of allergies – endocrinology • The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. • It represented the first time that hormone levels in the blood could be detected by an in vitro assay. Principle • Based on competition between unlabelled antigen and finite amount of corresponding labelled antigen for a limited number of antibody binding sites in a fixed amount of antiserum. • At equilibrium in the presence of an antigen excess there will be both free antigen antigen bound to antibody. • Under standard conditions the amount of labelled antigen bound to the antibody will decrease as the amount of unlabelled antigen in the sample increases. • 4Ag⃰ + 4 Ab → 4Ag⃰.Ab • 4 Ag + 4 Ag⃰ + 4 Ab → 2Ag⃰Ab + 2 AgAb + 2Ag⃰ + 2Ab • 12 Ag + 4 Ag⃰ + 4 Ab → Ag⃰ Ab + 3AgAb + 3Ag⃰ + 9 Ag • Principle: Uses an immune reaction • [Antigen – Antibody reaction] to estimate a ligand Ag + Ag* + Ab AgAb + Ag*Ab + Ag + Ab* – Unbound Ag* and Ag washed out – Radioactivity of bound residue measured – Ligand conc is inversely related to radioactivity [Ag : ligand to be measured ; Ag* radiolabelled ligand] • At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules. • The antibody-bound antigen is separated from the free antigen in the supernatant fluid, and the radioactivity of each is measured. Gamma Counter • From these data, a standard binding curve, like the one shown in red, can be drawn. • The samples to be assayed (the unknowns) are run in parallel. • After determining the ratio of bound to free antigen in each unknown, the antigen concentrations can be read directly from the standard curve. • The main drawbacks to radioimmunoassay are the expense and hazards if preparing and handling the radioactive antigen. • Both 125I or 131I emit gamma radiation that requires special counting equipment; • The body concentrates iodine atoms — radioactive or not — in the thyroid gland where they are incorporated in thyroxine (T4). • Despite these drawbacks, RIA has become a major tool in the clinical laboratory where it is used to assay • plasma levels of: – most of our hormones; – digitoxin or digoxin in patients receiving these drugs; – certain abused drugs • for the presence of hepatitis B surface antigen (HBsAg) in donated blood; • anti-DNA antibodies in systemic lupus erythematosus (SLE).