Double Immunodiffusion

Reference:

Ouchterlony, (1959).

Materials:

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.

Methods:

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 37^oC in a humid chamber.
5. The wells are filled with PBS, and the slides are incubated at 4^oC 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 37^oC, 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.