Double Immunodiffusion


Double Immunodiffusion (DID) is useful for the analysis of antigens and antibodies. This method is also known as Ouchterlony double diffusion, agar gel immunodiffusion or passive double immunodiffusion. In DID, both the antibody and antigen are allowed to diffuse into the gel. The antibodies react with specific antigens, forming large antigen-antibody complexes which can be observed as a line of precipitate. The pattern of the lines that form can determine whether the antigens are the same.


  • Agarose
  • Assay buffer
  • Glass plate
  • Gel punch
  • Template
  • Antiserum
  • Antigen solutions


  1. Dissolve 100 mg of agarose in 10 ml of assay buffer, by boiling to completely dissolve the agarose.
  2. Cool the solution to 55 °C, and pour agarose solution to a depth of 1 – 2 mm in a clean glass plate (petri dish or rectangular plate) placed on a horizontal surface.
  3. Note: Wipe the glass plates with alcohol and cotton to remove grease to ensure even spreading of agarose.
  4. Allow the gel to set for 30 minutes.
  5. Place the gel onto a template of the desired pattern and punch wells into the gel using a gel punch corresponding to the marks on the template.
  6. Note: Avoid forming rugged wells by using gentle suction.
  7. Fill wells with solutions of antigen and antiserum until the meniscus just disappears.
  8. Note: The concentration of antigen solution and the dilution of antiserum should be established by trial and error. See recommendations.
  9. Incubate the glass plate in a moist chamber overnight at 37 °C.
  10. Note: Ensure enough wet cotton is present to keep the chamber humid.
  11. Observe for opaque precipitant lines between the antiserum and antigen walls.


When using 5 µl samples, as a rough guide for undiluted antiserum the following concentration of antigen solutions should be used:

  • 1 mg/ml pure antigen
  • 50 mg/ml partially pure antigen
  • 500 mg/ml very impure antigen