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