1. Ouchterlony double diffusion
Bahiya Osrah

2. Introduction
Ouchterlony double diffusion is used to detect, identify, and quantify antibody and antigen
To test the similarity between antigens
Different geometrical patterns produced between antigen and antibody
To compare antigen
For disease diagnosis

3. Procedure Ag
Ab 2
Ab 1

4. Procedure 1.5g of agarose in 100ml of saline in conical flask
Heat in boiling water bath until the clumps are dissolved
Cool and then poor on petri dishes or microscopic slide cover
Allow the plates to cool
Cut out three wells and remove the gell plugs
Add the serum, antibody 1, antibody 2 each to different well
put the slide or the petridish in a bigger petri dish and use wet cottons or tissues to surround the petri
Cover
Incubate at 37C in moist chamber for overnight
Dye for 15 min
Remove dye 3 times each 4 min
Treat with 1% w/v tannic acid to aid visualization
Examine the pattern obtained and interpret the results

5. Filling Wells
50-60 µl

6. showing reagent levels
Cross section of wells
showing reagent levels
Over
Under
Correct
filled
filled

7. Hold Tip Vertical When
Filling Wells

8. Ag
Ab 2
Ab 1
Ab = anti-body
Ag = Antigen

9. RESULTS A C B A B C

10. Diffusion of Reagents Ag Ab Seen as a precipitin line
when concentrations are
optimal

11. Diffusion of Reagents Ag As
At 24 hours a
precipitin line is visible

12. Simple Immunodiffusion Reactions

13. Comments A B C
Antigens are identical and the Ab reacts with both antigen and gives
smooth line of precipitate
B. Antigens are different and the antibody reacts differently with antigens
C. Antibody reacts more with one antigen than the other