DID - Doubled Immunodiffusion

This method is also known as Ouchterlony double diffusion, agar gel immunodiffusion or passive double immunodiffusion.

The purpose of this method is to detect or measure antibodies and antigens through precipitation, which involves diffusing through agar or gel agarose.

Required Material:

• Agarose gel

• PBS

• Anti-serum and test antigens

• Incubator 37°C

• Weighing balance

• Gel punch

• Glass plate and conical flask measuring cylinder

Protocol:

1. Dissolve 100mg of agarose in 10ml of buffer and bring to a boil, agarose should be completely dissolved.

2. Cool the solution down to 55°C and pour agarose into a clean glass petri dish and allow to set for 30 minutes.

3. Using the gel puncher, punch out wells in the gel according to a template.

4. Fill the wells with the already prepared solution of antigen and anti-serum (Anti-serum is usually placed in the central well and different antigens are added to the wells surrounding the centre well).

5. Incubate the plate overnight at 37°C in a moist chamber.

Results:

Observe for opaque precipitant lines between the anti-serum and antigen wells to determine the antigen-antibody interaction.

If there are no precipitant lines this suggests an absence of reaction.

The possible results are:

• Full-Identity: This ‘arc’ pattern represents serological identity or the presence of a common epitope in antigens.

• Non-Identity: The crossed line pattern shows two separate reactions, which demonstrates the antigens are unrelated and share no common epitopes.

• Partial-Identity: This pattern shows that two antigens share a common epitope, however, some antibodies are not captured by the antigen, suggesting they are partially similar or cross-reactivity.