OUCHTERLONY DOUBLE IMMUNODIFFUSION • Double immunodiffusion is an agar gel immunodiffusion. • It is a special precipitation reaction on gels where antibodies react with specific antigens forming large antigen-antibody complexes which can be observed as a line of the precipitate. • In double immunodiffusion, both the antibody and antigen are allowed to diffuse into the gel. HISTORY • It was firstly discovered by Dr. Morris Goodman. • In 1948, Ouchterlony double diffusion technique was developed by Dr. Orjan Ouchterlony

1. ASMITA MUKHERJEE
(BSc Microbiology)

2. INTRODUCTION…
Immunodiffusion is a technique
used for studying reaction
between antibodies and
antigens by observing
precipitates formed by the
combination of specific antigen
and antibodies that have been
diffused in a gel in which they
have been separately placed.

3. HISTORY…
• It was firstly discovered by Dr. Morris
Goodman.
• In 1948, Ouchterlony double diffusion
technique was developed by Dr. Orjan
Ouchterlony

4. TYPES OF IMMUNODIFFUSION…
IMMUNODIFFUSION
Single Radial
Immunodiffusion
Double
Immunodiffusion

5. OUCHTERLONY
DOUBLE IMMUNODIFFUSION…
• Double immunodiffusion is an agar gel
immunodiffusion.
• It is a special precipitation reaction on gels where
antibodies react with specific antigens forming
large antigen-antibody complexes which can be
observed as a line of the precipitate.
• In double immunodiffusion, both the antibody and
antigen are allowed to diffuse into the gel.
• After application of the reactants in their
respective compartments, the antigen and the
antibody diffuse toward each other in the common
gel and a precipitate is formed at the place of
equivalence.

6. PRINCIPLE…
In Ouchterlony double immunodiffusion,
both antigen and antibody allowed to diffuse
into the gel. This assay is frequently used for
comparing different antigen preparations.
The method is called ‘Double’ since the
antigen and antibody are allowed to migrate
towards each other in a gel and a line of
precipitation is formed where the two
reactants meet. This precipitation is highly
specific and is used by people working with
diagnosis and protein detection technique.

7. • The pattern of lines that form can be interpreted to
determine whether the antigen are same or different as
illustrated below.
i. Pattern of identical – 1
ii. Pattern of non identical – 2
iii. Pattern of partially identical – 3
• Formation of lattice happens only when:
i. Antibody must be bivalent
ii. Antigen must be bivalent or polyvalent

8. MATERIALS REQUIRED…
• Chemicals: Agarose, Assay buffer, Anti-serum, Test
Antigen, pH 7.2, 0.05% Sodium azide
• Equipments: Incubator, Micro pipette, Tips, Weighing
balance, Gel punch with syringe
• Glass wares: Glass plate, Template, Conical flask,
Measuring cylinder
• Reagents: Alcohol, Distilled water

9. PROCEDURE…
1. Dissolve 100 mg of agarose in 10 ml of assay buffer, by
boiling to completely dissolve the agarose.
2. Cool the solution to 55 °C, and pour agarose solution to a
depth of 1 – 2 mm in a clean glass plate (petri dish or
rectangular plate) placed on a horizontal surface.
(Note: Wipe the glass plates with alcohol and cotton to
remove grease to ensure even spreading of agarose.)
3. Allow the gel to set for 30 minutes.
4. Place the gel onto a template of the desired pattern and
punch wells into the gel using a gel punch corresponding
to the marks on the template. (Note: Avoid forming
rugged wells by using gentle suction.)

10. 5. Fill wells with solutions of antigen and antiserum
until the meniscus just disappears. (Note: The
concentration of antigen solution and the dilution of
antiserum should be established by trial and
error. See recommendations.)
6. Incubate the glass plate in a moist chamber
overnight at 37 °C. (Note: Ensure enough wet
cotton is present to keep the chamber humid.)
7. Observe for opaque precipitant lines between the
antiserum and antigen walls.

11. INTERPRETATION…
• A full identity (i.e. a continuous line): Line of precipitation
at their junction forming an arc represents serologic
identity or the presence of a common epitope in antigens.
• Non-identity (i.e. the two lines cross completely): A
pattern of crossed lines demonstrates two separate
reactions and indicates that the compared antigens are
unrelated and share no common epitopes.
• Partial identity (i.e. a continuous line with a spur at one
end): The two antigens share a common epitope, but
some antibody molecules are not captured by the antigen
and traverse through the initial precipitin line to combine
with additional epitopes found in the more complex
antigen.

13. ZONAL REACTION…
• Post zone reaction
• Pre zone reaction
• Equivalence zone reaction

14. DOUBLE DIFFUSION RESULT…
• The presence of an opaque precipitant line between
the antiserum and antigen wells indicates antigen-
antibody interaction.
• Absence of precipitant line suggests the absence of
reaction.
• When more than one well is used there are many
possible outcomes based on the reactivity of the
antigen and antibody selected.

15. EXAMPLES…
• Home pregnancy test
i. It is a modification of an agglutination
experiment.
ii. It is called agglutination inhibition.
iii. It is good because it is sensitive to small
amount of antigen

17. APPLICATION…
• It is useful for the analysis of antigens and
antibodies.
• It is used in the detection, identification, and
qualification of antibodies and antigens, such
as immunoglobins and extractable nuclear
antigens.
• Demonstration of antibodies in serodiagnosis
of smallpox.
• Identification of fungal antigens.

18. REFERANCE…
• https://www.slideshare.net/msaltyy/double-
immunodiffusion-technique
• https://microbenotes.com/ouchterlony-double-
immunodiffusion-technique/
• https://www.abbexa.com/double-
immunodiffusion