VMC 221: Veterinary Immunology and Serology (1+1)

RADIO IMMUNO ASSAY(RIA)

• It is one of the most sensitive techniques for detecting antigen or antibody. RIA was first developed in the 1960s by two   endocrinologists   S. A. Berson and Rosalyn Yalow to determine levels of insulin-anti insulin complexes in diabetics.
• RIA for antigen detection
  • Competitive RIA are based on the principle that unlabelled antigen will displace radio labeled antigen from immune complexes.
  • It is extremely sensitive and commonly used to detect trace amounts of drugs or antigen.
  • The antigen or drug is labeled with an isotope such as tritium, C-14 or I -125. When radio labeled antigen is mixed with its specific antibody it combines to form immune complexes that may be precipitated out of solution.
  • The radioactivity of the supernatant fluid is a measure of the amount of unbound antigen.
  • If unlabelled antigen is added to the mixture of labeled antigen and unbound antibody, it will compete with the labeled antigen for antibody binding sites.
  • As a result, some labeled antigen will be unable to bind antibodies and the radioactivity in the supernatant will increase.
  • If a standard curve is plotted using known amounts of unlabelled antigen, then the amount of antigen in a test sample can be measured by referring to this standard curve.
• RIA for antibody detection
  • In this technique antigen impregnated cellulose discs are immersed in test serum so that specific antibody binds to the antigen.
  • Unbound antibody is removed by washing and the disc is immersed in radiolabeled antiglobulin.
  • The antiglobulin binds to the disk only if antibodies have first bound to the antigen.
  • The amount of radioactivity bound to the disk is a measure of the level of antibody activity.
  • The cost of the equipment and the health hazard of working with radioisotopes argue against its use in small laboratories.
  • The radio allergosorbant test (RAST) is a primary binding test used to measure levels of specific IgE in allergic animals. Here the allergen is coated on a solid phase and IgE against that allergen only is detected. In radio immunosorbant test (RIST), which is used to detect serum levels of total IgE, anti IgE is coupled to a solid phase and is incubated in patient serum (figure).