UNIT 10.24 Solution Radioimmunoassay of Proteins and Peptides
Published Online: 1 NOV 2005
Copyright © 2005 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Smith, J. A. 2005. Solution Radioimmunoassay of Proteins and Peptides. Current Protocols in Molecular Biology. 10:VI:10.24.
- Published Online: 1 NOV 2005
- Published Print: OCT 2005
This is not the most recent version of the article. View current version (1 MAY 2006)
This unit deals with solution radioimmunoassays (RIAs), using the double antibody/polyethylene glycol (PEG) method. A solution RIA determines the amount of a specific protein (or peptide) in an unknown sample by comparison to a standard curve developed using known amounts of the same protein (or peptide) (i.e., so-called standards). The amount of protein (or peptide) antigen in an unknown sample is determined based on its ability to compete with a radiolabeled protein (or peptide) (i.e., so-called tracer) for binding to the primary anti-protein (or anti-peptide) antibody. A secondary antibody directed at the primary antibody in the presence of PEG is used to precipitate the primary antibody-protein (or protein) complexes. The amount of radioactivity in the complex is inversely proportional to the amount of protein in the unknown sample, since there is competition between the radiolabeled tracer and the unlabeled protein (or peptide) in the unknown. Under ideal conditions, an RIA is capable of measuring picograms (10−12 g) of a protein (or peptide) antigen, but only if high specific activity radiolabeling of the standard protein (or peptide) can be achieved. Radioiodine (either 125I or 131I) is the label of choice, because of the significant increase in radioactive disintegrations per unit time (in comparison to 3H or 14C).
- anti-protein (or peptide antiserum);
- double-antibody method;
- γ counter;
- homologous RIA;
- polyethylene glycol;
- primary antibody;
- protein (or peptide) standards;
- protein (or peptide) tracer;
- quality controls;
- secondary antibody;
- solution RNA