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Ouchterlony Double Diffusion - Patterns


Materials Required:


  • Agarose
  • 1X Assay buffer
  • Antiserum
  • Test Antigens (Ag1 and Ag2)
  • Glass slide
  • Gel punch with syringe
  • Template
  • Incubator (37˚C)
  • Conical flask
  • Measuring cylinder
  • Alcohol
  • Distilled water
  • Micropipette and pipette tip
  • Petri plate
  • Cotton




  • Prepare 25 ml of 1.2% agarose (0.3 g /25 ml) in 1X assay buffer by boiling to dissolve the agarose completely.
  • Cool the solution to 55-60°C and pour 4 ml/slide on to  grease free glass slide placed on a horizontal surface. Allow the gel to set for 30 minutes.
  • Punch wells by keeping the glass plate on the template.
  • Fill the lower well with 10µl of antiserum and the upper two wells with 10 µl each of Antigen 1 and 2
  • Keep the glass plate in a moist chamber overnight at 37°C.
  • After incubation, observe for opaque precipitin lines between the antigen and antisera wells.


Observations and Results:


Observe for the presence of precipitin lines between the antigen and antisera wells.

  • If pattern A or ‘pattern of identity’ is observed between the antigens and the antiserum, it indicates that the antigens are immunologically identical.
  •  If pattern B or ‘pattern of partial identity’ is observed, it indicates that the antigens are partially similar or cross-reactive.
  •  If pattern C or ‘pattern of non-identity’ is observed, it indicates that there is no cross-reaction between the antigens. i.e., the two antigens are immunologically unrelated.


 Fig 2 : Glass plate showing pattern of lines obtained following Ouchterlony double diffusion


Things to watch out for:


  • Wipe the glass plate with alcohol to make it grease free for even spreading of agarose.
  • Ensure that the moist chamber has enough wet cotton to keep the chamber humid.
  • If you are provided with 10X assay buffer, dilute the required amount of 10X assay buffer to 1X with distilled water.
  • Reconstitute each vial containing the antigen and antiserum with 0.2 ml of 1X assay buffer. Mix it well and allow it to stand for 30 minutes. Store at 4°C.  Use within 3 months.
  • Assay Buffer: Phosphate buffered saline (PBS)

Wear heat protective gloves when making the agarose solution



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