Solution Radioimmunoassay of Proteins and Peptides

John A. Smith1

1 University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.24
DOI:  10.1002/0471142727.mb1024s74
Online Posting Date:  May, 2006
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Abstract

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.

Keywords: antibody; anti-protein antiserum; double-antibody method; RIA; peptides; polyethylene glycol; proteins; radioimmunoassay

     
 
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Table of Contents

  • Safety with Radioactive Iodine
  • Basic Protocol 1: Solution Radioimmunoassay for Proteins and Peptides
  • Support Protocol 1: Iodination of Protein Tracer Using the Bolton‐Hunter Reagent
  • Support Protocol 2: Iodination of Protein Tracer by Lactoperoxidase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Solution Radioimmunoassay for Proteins and Peptides

  Materials
  • Assay buffer (see recipe)
  • Protein standards (see recipe)
  • Protein quality controls 1 and 2 (see recipe)
  • Unknown samples (e.g., plasma, serum, tissue culture medium)
  • Protein tracer, iodinated with 125I (see recipe)
  • Protein tracer buffer (see recipe)
  • Anti‐protein antiserum (containing primary antibody; see recipe)
  • Precipitating secondary antibody/PEG solution (see recipe)
  • Assay tubes: 12 × 75–cm glass test tubes (polypropylene or polyethylene tubes may be used provided that the pellets remain compacted following the decanting step)
  • Refrigerated centrifuge with swinging‐bucket rotor
  • γ counter
  • Log/log graph paper

Support Protocol 1: Iodination of Protein Tracer Using the Bolton‐Hunter Reagent

  Materials
  • ∼100 mCi/ml Na125I (IMS30, GE Healthcare Life Sciences; sp. act. >0.6 TBq/mg iodide)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.5 mg/ml Bolton‐Hunter reagent (Pierce cat. no. 27710) in dimethylsulfoxide (DMSO)
  • Chloramine‐T solution: dissolve 1 mg chloramine‐T in 250 to 500 µl of PBS just before use
  • Dimethylformamide
  • Benzene
  • Purified protein of interest (i.e., protein tracer; see protocol 1)
  • Sodium borate buffer (see recipe), cold
  • 1.5‐ml screw‐cap microcentrifuge tubes
  • γ counter
  • Additional reagents and equipment for gel‐filtration chromatography (unit 10.9), dialysis ( appendix 3C), or ultrafiltration ( appendix 3C)

Support Protocol 2: Iodination of Protein Tracer by Lactoperoxidase

  Materials
  • Purified protein of interest (i.e., protein tracer; see protocol 1)
  • Phosphate‐buffered saline (PBS; see recipe)
  • ∼100 mCi/ml Na125I (IMS30, GE Healthcare Life Sciences; sp. act. >0.6 TBq/mg iodide)
  • Lactoperoxidase solution: dissolve 1.5 mg of lactoperoxidase (from bovine milk; Sigma‐Aldrich or Worthington) in 1 ml of PBS
  • Hydrogen peroxide solution (see recipe)
  • 1.5‐ml screw‐cap microcentrifuge tubes
  • Additional reagents and equipment for purifying and concentrating the 125I‐labeled protein tracer ( protocol 2)
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Figures

Videos

Literature Cited

   Bolton, A.E. and Hunter, W.M. 1973. The labeling of proteins to highly specific radioactivities by conjugation to a 125I‐containing acylating agent. Biochem. J. 133:529‐539.
   Feitelson, M.A., Wettstein, F.O, and Stevens, J.G. 1981. Tryptic peptide mapping of picomolar quantities of protein labeled with the Bolton‐Hunter reagent. Anal. Biochem. 116:473‐479.
   Hunter, W.M. 1973. Radioimmunoassay. In Handbook of Experimental Immunology, Immunochemistry (D.M. Weir, ed.), Vol. 1, pp. 17.1‐17.36. Blackwell Scientific Publications, Oxford.
   Hunter, W.M. and Greenwood, F.C. 1962. Preparation of iodine‐131 labeled human growth hormone of high specific activity. Nature 194:495‐496.
   Marchalonis, J.J. 1969. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem. J. 113:299‐305.
   Re, R.N. and Haber, E. 1980. Radioimmunoassay. In Methods in Immunodiagnosis (N.R. Rose and P.E. Bigazzi, eds.) pp. 147‐160. John Wiley & Sons, New York.
   Salacinski, P.R.P., McLean, C., Sykes, J., Clement‐Jones, V., and Lowry, P. 1981. Iodination of proteins, glycoproteins, and peptides using a solid phase oxidation reagent, 1,3,4,6‐tetrachloro‐3α,6α‐diphenylglycouril (Iodogen). Anal. Biochem. 117:136‐146.
   Wilbur, D.S., Hadley, S.W., Hylarides, M.D., Abrams, P.G., Beaumier, P.A., Morgan, A.C., Reno, J.M., and Fritzberg, A.R. 1989. Development of a stable radioiodinating reagent to label monoclonal antibodies for radiotherapy of cancer. J. Nucl. Med. 30:216‐226.
   Yalow, R.S. and Berson, S.A. 1960. Immunoassay of endogenous plasma insulin in man. J. Clin. Invest. 39:1157‐1175.
Key References
   Bolton‐Hunter Reagent Instructions, Pierce Chemical Company, Rockford, Ill.
  This reference was used as the basis for the method in this unit.
   Hunter, 1973. See above.
  Excellent review of all aspects of radioimmuno‐assay development.
   Marchalonis, 1969. See above.
  This paper describes the use of catalytic amounts of lactoperoxidase to iodinate proteins gently and with high specific activity.
   Yalow and Berson, 1960. See above.
  This paper describes the development of the first radioimmunoassay and its use to measure insulin in human plasma. Dr. Rosalind Yalow was awarded the 1977 Nobel Prize in Physiology or Medicine for the invention of this method.
Internet Resources
   http://www.cyfc.psu.edu/funding/L2_dr2.html
  This URL presents an excellent assay development plan for measuring biomarkers by immunological assays (e.g., RIAs and EIAs). It discusses feasibility considerations and internal and external validation, as well as aspects of sample selection, collection, handling, and storage.
   http://www.lincoresearch.com/products/pl‐84k.html
  This URL provides access to a detailed protocol for a commercially available primate leptin RIA, developed by LINCO Research. This protocol is an excellent example of the use of a double‐antibody/PEG method for doing RIAs. This protocol was used as the basis for the protocol in this unit.
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