This unit describes a solution-phase radioimmunoassay (RIA) using the double antibody/PEG method. A solution RIA determines the amount of a specific protein (or peptide) in a sample by comparison to a standard curve of the same protein (or peptide). The amount of protein (or peptide) in the sample is determined based on its ability to compete with a radiolabeled tracer for binding to a specific antibody. A secondary antibody and PEG are used to precipitate the primary antibody-protein complexes. The amount of radioactivity in the complex is inversely proportional to the amount of protein in the sample. Under ideal conditions, an RIA is capable of measuring picograms of a protein (or peptide), but only if the tracer is labeled at a high specific activity. To this end, this unit also presents protocols for radioiodination of proteins and peptides using lactoperoxidase or the Bolton-Hunter reagent.
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