AIM: To study Ouchterlony’s immuno – double diffusion method.

Requirements:
Agarose (1.2% in assay buffer),
Commercial antigen and antiserum,
Assay buffer (1x PBS), Distilled water, Eosin, Methylene blue,
slide, Gel plunger, filter paper, Dropper, Glass stirrer, Beaker,
Micropipette, Moist chamber.
Principle:
• Precipitation reactions are based on the interaction of antibodies and
antigens. They are based on two soluble reactants that come together to
make one insoluble product, the precipitate.
• These reactions depend on the formation of lattices (cross-links) when
antigen and antibody exist in optimal proportions.
• Excess of either component reduces lattice formation and subsequent
precipitation.
• Precipitation reactions differ from agglutination reactions in the size and
solubility of the antigen and sensitivity.
• Antigens are soluble molecules and larger in size in precipitation reactions.
• Precipitation methods include double immunodiffusion (qualitative gel
technique that determines the relationship between antigen and
antibody), radial immunodiffusion (semi-quantitation of proteins by gel
diffusion using antibody incorporated in agar), and electro-
immunodiffusion (variation of the double immunodiffusion method
reaction that uses an electric current to enhance the mobility of the
reactants toward each other).
Immuno-diffusion is a technique for detecting or measuring antibodies and
antigens by their precipitation when diffused together through a gel or other
medium.
The commonly known types are
1. Single diffusion in one dimension (Oudin procedure)
2. Double diffusion in one dimension (Oakley Fulthorpe procedure)
3. Single diffusion in two dimension (radial immunodiffusion or Mancini
method)
4. Double diffusion in two dimensions (Ouchterlony double immunodiffusion)
Double immuno-diffusion of antigen and antibody was first described by Örjan
Ouchterlony (1948).
It is also known as agar gel immunodiffusion or passive double
immunodiffusion).
It is an immunological technique used in the detection, identification and
quantification of antibodies and antigens, such as immunoglobulins and
extractable nuclear antigens.
• In Ouchterlony double diffusion, both antigen and antibody allowed to
diffuse in to 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 are meeting at the zone of
equivalence.
• This precipitation is highly specific and is used by people working with
diagnosis and protein detection technique.
The pattern of lines that form can be interpreted to determine whether the
antigens are same or different.
Total identity – 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.
Partial Identity - In the ‘pattern of partial identity’, the antibodies in the
antiserum react more with one of the antigens than the other. The ‘spur’ is
thought to result from the determinants present in one antigen but lacking in
the other antigen.
Non-identity - 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.
Different patterns of lines obtained on Ouchterlony double diffusion
 Ouchterlony double diffusion - Partial
Ouchterlony double diffusion - Total identity of antigens
identity of antigens
PROCEDURE:
• Prepare 10 ml of 1.2% agarose (1.2 g /100 ml) in 1X assay buffer by boiling to
dissolve the agarose completely.
• Cool the solution to 55-60°C and pour 3 - 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. To demonstrate arc formation only two wells are made
and eosin and methylene blue added to the wells separately.
• 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.
OBSERVATION AND RESULTS:
• Precipitation lines were observed between antigen and antisera wells. The
arc showed partial identity of antigens.
• In another slide, the precipitation line was formed between Eosin and
Methylene blue dye.

Paste your own

result

Arc formation between Eosin and methyline blue dye

DISCUSSION:
• 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 antibody is contained in the precipitate. This is called the
antibody-excess zone (i.e. prozone phenomenon).
• As more antigens are added, the amount of 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.

PRECAUTION:
• Wipe the glass plate with alcohol to make it grease free for even spreading
of agarose.
• Spreading of agarose on slide should be uniform.
• The well should be carefully formed avoiding rugged walls.
• The amount of antigen and antibody should be carefully added without
spilling or mixing.
• Ensure that the moist chamber has enough wet cotton to keep the chamber
humid.