Double Immunodiffusion

Categorized: Immunology Based Methods


Ouchterlony, (1959).


0.08% barbitone buffer:

  1. 12.0 g sodium 5’5 diethyl barbiturate
  2. 4.40 g 5’5 diethylbarbituric acid
  3. Adjust the pH to 8.2 with 1.0 M NaOH and make up to 1 litre with distilled water.


  1. Prepare a 2% (w/v) agarose (Standard low Mr agarose, Biorad) suspension in 0.08% barbitone buffer.
  2. Add Triton X-100 to a concentration of 1% (v/v) and pour 8ml of the agarose suspension onto a glass slides.
  3. The outer antigen wells are filled with 20ug of the solubilized antigen and the centre antiserum well is filled with undiluted or two-fold dilutions of antiserum.
  4. The agarose slides are incubated for 16 hours at 37oC in a humid chamber.
  5. The wells are filled with PBS, and the slides are incubated at 4oC for a further 72 hours.
  6. The slides are pressed dry under a stack of Whatman 3M filter papers, washed five times in PBS for 45 minutes at 37oC, dried again, stained in 1% (w/v) Coomassie brilliant blue dissolved in absolute methanol, acetic acid and water at a ratio of 4:1:4 and destained in the same solution without the dye.