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Matrix No. : MEB080004


1. To learn the proper technique of Oucheterlony double diffusion.
2. To learn how to interpret the results pbtained from Oucheterlony double

Ouchterlony or double immune diffusion is a method where the antigen
(from sample) and antibody (known concentration) are placed respectively into 2
neighboring wells punched in the agar. Each reactants will diffuse out radially of
their respectively wells. The concentration of the reactants is inversely
proportional to the distances from the well. Immuno precipitate will form when
antigen and antibody meet in optimal proportions. There are two factors that
determine location of the precipitin band which are the concentration and the
molecular size of the reactants.
Precipitation occurs with most antigens because the antigen is multivalent
(i.e. has several antigenic determinants per molecule to which antibodies can
bind). Antibodies have at least two antigen binding sites (and in the case of IgM
there is a multimeric complex with up to 10 antigen binding sites), thus large
aggregates or gel-like lattices of antigen and antibody are formed.
Experimentally, an increasing amount of antigen is added to a constant amount
of antibody in solution, initially at low antigen concentration, all of the antigen is
contained in the precipitate. This is called the antibody-excess zone (i.e. prozone
phenomenon). As more antigen is added, the amount protein precipitated
increases until the antigen/antibody molecules are at an optimal ratio. This is
known as the zone of equivalence or equivalence point. When the amount of
antigen in solution exceeds the amount of antibody, the amount of precipitation
will decrease. This is known as the antigen excess zone.

1. Three test antiserum (antiserum A, B and C)
2. Corresponding antigens (Ag A1, A2, B1, B2, C1 and C2)
3. 25ml of 1.2% agarose (0.3g/ 10ml) in 1 x assay buffer
4. Gel slide with template
5. Gel punch with syringe

1. 25mL of 1.2% agarose (0.3/25mL) in 1x assay buffer was prepared by
boiling to dissolve the agaorse completely.
(Note: 5 plates was prepared at a time and used on the same day)
2. The solution was cooled to 55-60°C and poured 4mL/plate onto 5 grease
free glass plates placed on a horizontal surface. The gel was allowed to set
for 30 minutes.
3. Wells were punched by keeping the glass plate on the template.
4. The wells were filled with 10µL each of the antiserum and the
corresponding antigens as shown below:

Antiserum A Antiserum B Antiserum C

Ag A1 Ag A2 Ag B1 Ag B2 Ag C1 Ag C2

Pattern of addition of antigen and antiserum to the wells.

5. The glass were kept in a moist chamber overnight at 37°C.

6. After incubation, the glass plates were observed for opaque precipitin lines
between the antigen and antisera wells.


Antiserum A Antiserum B Antiserum C

Ag A1 Ag A2 Ag B1 Ag B2 Ag C1 Ag C2


1. Ouchterlony (immuno-double diffusion) allows one to determine

relationships of different antigens, qualitatively and quantitatively. A well
filled with antigen in an agar plate will react with wells filled with antibody.
As the reactants diffuse towards one another, a line of precipitate will form
at optimum concentrations.

2. The percentage of precipitation will decrease when either one of them is in

excess. The location of this precipitin line formed depends on two factors
which are:
a. The molecular size of the reactants
b. The concentration of the reactants

3. The concentrations of the antigen and antiserum are relatively higher near
their respective wells. As they diffuse further from the wells, their
concentrations become equivalent and the antigen-antiserum complex
precipitates to form a precipitin line. This precipitin formed depends on
a. the distance between wells
b. the concentration of the gel displayed
c. the thickness of the gel displayed
d. the viscosity of the gel displayed

4. The precipitation patterns on the Ouchterlony plates can be interpreted

a. Pattern of Identity: A
The antibodies in the antiserum react with both the antigens
resulting in a smooth line of precipitate. The antibodies cannot
distinguish between the two antigens i.e. the two antigens are
immunologically identical.
b. Pattern of Partial Identity: B
In the pattern of partial identity, the antibodies in the antiserum
react more with one of the antigens (the t diffuses from the left
hand well in the figure) than the other. The ‘spur’ is thought to
result from the determinants present in one antigen but lacking in

the other antigen.

c. Pattern of Non-Identity: C
In the ‘pattern of non-identity’, none of the antibodies in the
antiserum react with antigenic determinants that may be present in
both the antigens i.e. the two antigens are immunologically
unrelated as far as that antiserum is concerned.

5. Inaccuracy of results may due to:

a. Contaminations of the gel or reactants and the present of absorbent
may give unnecessary precipitin lines
b. The materials used not kept properly at 4oC and may be kept too
c. Other precipitin especially non-antigen-antibody precipitation may
occur on the gel if preparation not thoroughly.

Ouchterlony double diffusion is a useful technique used to determine the
presence and compare the amount of antigen-antiserum interaction that occurs.