Radioimmunoassay

• Chemiluminescence


  The emission of light caused by a chemical reaction (typically oxidation) producing an excited molecule that decays back to its original ground state
• Most common chemiluminescent substances

  • Luminol
  • Ruthenium derivatives
  • Acridinium esters
  • Nitrophenyl oxalates
• Homogeneous assay


  No washing step involved
• Heterogeneous assay


  Needs washing step
• Chemiluminescence is versatile because we can detect antigen, antibody, hormones and enzymes
• Solid phase


  • Magnetic Particles
  • Microtiter Plates
  • Gels in slide
• Chemiluminescent immunoassay principle


  1. Antigen attaches to well

  2. Add antibody

  3. Add enzyme substrate

  4. Add chemiluminescent substance
• Indirect chemiluminescent immunoassay


  We have a primary antibody and a secondary antibody, the primary antibody is from the serum sample and the secondary antibody is the reagent
• In nature, bioluminescent substances like fireflies emit light due to oxidation
• Luminometers


  • Automated equipment
  • Can adjust color of light emitted
  • Can run multiple specimens and tests
  • More expensive than other equipment
• Advantages of chemiluminescent immunoassay

  • Excellent sensitivity
  • Reagents are stable and relatively non-toxic
  • Inexpensive to perform
  • Faster turnaround time
• Disadvantages of chemiluminescent immunoassay

  • False results from lack of precision in injection of hydrogen peroxide
  • Biological materials like plasma or urine can cause quenching of light emission
• Radioimmunoassay (RIA)


  Technique in which a radioisotope is used as a tag or label for the detection of antigen-antibody complexes
• Radioisotopes are covalently attached to antigens or antibodies, and the radiation emitted can be measured by a gamma counter
• The most popular radioisotope used in RIA is 125I
• RIA was developed in 1959 by endocrinologist Rosalyn Yalow and Solomon Berson, and Yalow was awarded the Nobel Prize for Medicine in 1977
• Competitive binding principle of RIA

  The analyte being detected competes with a radiolabeled analyte for a limited number of binding sites on a high affinity antibody
• RIA detects the presence of antigen
• Relationship between amount of label in bound phase and amount of patient antigen

  Indirectly proportional
• Steps in generating a standard curve for RIA

  1. Measure radioactivity using fixed excess concentration of radiolabeled antigen and varying known concentration of unlabeled antigen

  2. Radioactivity will decrease as concentration of unlabeled antigen increases
• As the concentration of unlabeled antigen increases, the radioactivity decreases
• Radioimmunoassay instrumentation includes a scintillation counter that measures ionizing radiation
• The smallest amount of antibody that can be detected by radioimmunoassay is 0.0006-0.006