Ouchterlony double diffusion Bahiya Osrah

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

Procedure http://www.youtube.com/watch?v=hmK7yYr2T54 Ag Ab 2 Ab 1

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

Filling Wells 50-60 µl

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

Hold Tip Vertical When Filling Wells

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

RESULTS A C B A B C

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

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

Simple Immunodiffusion Reactions

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