Immunological techniques-IMMUNODIFFUSION

PATHOLOGY
AND
MICROBIOLOGY
PRESENTED BY,
K.K.LEKA.,ME,
ASSISTANT
PROFESSOR,
DEPARTMENT OF BIOMEDICAL
ENGINEERING, ESEC

IMMUNODIFFUSION
• The precipitation test in a agar gel is termed as immunodiffusion test.
• In this test, reactant are added to the gel and antigen-antibody combination
occurs by the means of diffusion.
• The rate of diffusion is affected by the size of the particle, temperature, gel
viscosity, amount of hydration and interaction between the matrix and
reactants.
• An agar concentration of 0.3-1.5% allows for diffusion of the most of the
reactants.
• One of the major advantages of immunodiffusion reaction is that the line of
precipitation is visible as a band which can also be stained for preservation.
• Another advantage is that it can be used to detect, identify the cross reaction and
non-identify between different antigens in a reacting mixture.

TYPES OF IMMUNODIFFUSION REACTION
Immunodiffusion reaction are classified based on the ;
Number of reactant diffusing and Direction of diffusion,
as follows;
❑ Single diffusion in one dimension
❑ Single diffusion in two dimension
❑ Double diffusion in one dimension
❑ Double diffusion in two dimension

Single diffusion in one dimension
• In this method, antibody is
incorporated into agar gel in a
test tube and the antigen solution
is poured over it.
• During the course of time, the
antibody in agar gel and a line
of precipitation is formed.
• The number of precipitate band
shows the number of different
antigen present in antigen
solution.

Single diffusion in two dimension
• It is also called radial immunodiffusion.
• In this method, antiserum solution containing antibody is incorporated
in agar gel on a slide or petridish. The wells are cut on the surface of
the gel.
• The antigen is then applied to the well cut into the gel. When antibody
already present in gel reacts with antigen which diffuses out of the well,
a ring of precipitation is formed around the wells.
• The diameter of the ring is directly proportional to the concentration
of the antigen.
• Radial immunodiffusion has been used for the quantitative estimation of
antibody and antigen in serum.

Double diffusion in one dimension
• In this method, the antibody is incorporated in agar gel in a test
tube above which a layer of plain agar is placed.
• The antigen is then layered on the top of this plain.
• During the course of time, the antigen and antibody move
toward each other through the intervening layer of plain agar.
• In this zone of plain agar, both antigen and antibody react
with each other to form a band of precipitation at their
optimum concentration.

Double diffusion in two dimension
• In this method, both the antigen and antibody diffuse
independently through agar gel in two dimensions, horizontally
and vertically.
• The test is performed by cutting wells in the agar gel poured on a
glass slide or in a petri dish.
• The antiserum consisting of antibody is placed in the central well
and different antigens are added to the wells surrounding the
center well.
• After an incubation period of 12-48 hours in a moist chamber,
the lines of precipitin are formed at the sites of combination of
antigen and antibody.

IMMUNOELECTROPHORESIS
• Immunoelectrophoresis refers to precipitation in agar under an
electric field.
• It is a process of a combination of immuno-diffusion and
electrophoresis.
• An antigen mixture is first separated into its component parts by
electrophoresis and then tested by double immuno-diffusion.
• Antigens are placed into wells cut in a gel (without antibody) and
electrophoresed. A trough is then cut in the gel into which antibodies
are placed.
• The antibodies diffuse laterally to meet diffusing antigens, and
lattice formation and precipitation occur permitting determination
of the nature of the antigens.

• When an electric current is applied to a slide layered with gel, the
antigen mixture placed in wells is separated into individual antigen
components according to their charge and size.
• Following electrophoresis, the separated antigens are reacted with
specific antisera placed in troughs parallel to the electrophoretic
migration and diffusion is allowed to occur.
• Antiserum present in the trough moves toward the antigen
components resulting in the formation of separate precipitin lines
in 18-24 hrs, each indicating reaction between individual proteins
with its antibody.

Result
• The presence of elliptical precipitin arcs represents antigen-antibody
interaction.
• The absence of the formation of precipitate suggests no reaction.
• Different antigens (proteins) can be identified based on the intensity,
shape, and position of the precipitation lines.
Applications
• The test helps in the identification and approximate quantization of
various proteins present in the serum. Immunoelectrophoresis created
a breakthrough in protein identification and in immunology.
• This method is useful to monitor antigen and antigen-antibody purity and
to identify a single antigen in a mixture of antigens.

RADIOIMMUNOASSAY
(RIA)
• Radioimmunoassay (RIA) is an immunological assay to analyze any
antigen in the sample with high sensitivity. Usually, with
Radioimmunoassay any biological sample for which a specific antibody
exists can be determined.
• The best advantage of radioimmunoassay is that you do not need the
samples or substances in higher quantities and concentrations, even
minute level of sample size is accepted. Radioimmunoassay is the most
specific and sensitive type of immunoassays available.
• Basically, radioimmunoassay is based on three principles which give it
high sensitivity.
❑ A strong immune binding reaction: Antigen vs Antibody reaction
❑ The competitive binding reaction which gives specificity

Radioimmunoassay Method
• In the basic method of Radioimmunoassay, we use the target antigen
which is labeled radioactively and bound to its specific antibodies.
• We will require a limited and known antibody to be added in a specific
amount in Radioimmunoassay.
• A sample is then added in order to initiate a reaction competitive in
nature, of the labeled antigens from the preparation, and the unlabeled
antigens from the sample, with the specific antibodies.
• This reaction to the antibodies will release a certain amount of labeled
antigen.
• This amount is correlative to the ratio of labeled to unlabeled antigen.

Uses of Radioimmunoassay (RIA)
• IA has revolutionized research and clinical practice specially
in blood banking, diagnosis of allergy and endocrinology.
Furthermore it is used to:
❖ The test can be used to determine very small quantities
(e.g. nanogram) of antigens and antibodies in the serum.
❖ The test is used for quantitation of hormones, drugs,
HBsAg, and other viral antigens.
❖ Analyze nanomolar and picomolar concentrations of
hormones in biological fluids.

Immunological techniques-IMMUNODIFFUSION

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Immunological techniques-IMMUNODIFFUSION