UNIVERSITY INSTITUTE OF

ENGINEERING-DEPARTMENT OF
BIOTECHNOLOGY ENGINEERING

Bachelor of Engineering (Biotechnology Engineering)

Subject Name : Immunology
Code : 21BEH-223

DISCOVER . LEARN . EMPOWER

Chapter 3 Techniques in immunology: Identification and measurement of antibodies and antigens,
Agglutination and precipitation reactions, Radial Immunodiffusion, Immunoelectrophoresis,
Radio Immunoassay, ELISA, Western blot, Immunofluorescence, Lateral flow assay, active
and passive immunization, types of vaccines, edible vaccines
 

2
Methods of Ag-Ab interaction
• Agglutination
• Precipitation
• Hemagglutination
• Radial-immunoassays
• ELISA
• Immunofluorescence
• Immunoblotting
• Lateral Flow assay
Agglutination reaction

• When a particular antigen is mixed with its antibody in the presence of

electrolytes at a suitable temperature and pH, the particles are clumped or
agglutinated. This process is called agglutination.

• In other word, it is an antigen-antibody reaction in which a particulate antigen

combines with its antibody, resulting in formation of visible clumping of particles.

• Agglutination tests are widely used in the clinical and diagnostic laboratories

because they are simple to perform, highly specific, inexpensive, rapid and
reasonably sensitive.
• It occurs optimally when the antigens and antibody reacts in
equivalent proportions.

• Incomplete or monovalent antibodies do not cause agglutination,

though they combine with the antigen.

• Antibodies that produce such reactions are called agglutinins.

Examples of Agglutination test

• Slide agglutination test

• Plate agglutination test
• Tube agglutination test
• Microscopic agglutination test
• Hemagglutination test (HAT)
Agglutination is used in blood typing
• Agglutination is a process in which particles, such as red blood cells, clump
together in response to the presence of certain antibodies.
• It is used in blood typing to determine a person's blood type based on the
presence or absence of specific antigens on the surface of red blood cells and
the corresponding antibodies in their blood plasma.
• In blood typing, a sample of blood is mixed with antibodies that are specific to
certain blood types.
• If the corresponding antigen is present on the red blood cells, the antibodies
will bind to them, causing agglutination, or clumping of the cells.
• The type of blood can be determined by observing which antibodies cause
agglutination.
• For example, if a person has type A blood, their red blood cells have the A
antigen on their surface. When their blood is mixed with anti-A antibodies,
the antibodies will bind to the A antigen, causing agglutination.

• Similarly, if a person has type B blood, their red blood cells have the B
antigen on their surface. When their blood is mixed with anti-B antibodies,
the antibodies will bind to the B antigen, causing agglutination.
• If a person has type AB blood, their red blood cells have both the A and B antigens on
their surface. When their blood is mixed with either anti-A or anti-B antibodies, the
corresponding antibodies will bind to the respective antigens, causing agglutination.

• If a person has type O blood, their red blood cells do not have either the A or B
antigen on their surface. When their blood is mixed with both anti-A and anti-B
antibodies, there will be no agglutination because there are no corresponding
antigens present.
• Agglutination is a crucial step in blood typing as it allows for the accurate
identification of blood types, which is essential in blood transfusions and
other medical procedures where matching blood types is critical to ensure
safe and effective treatment.
Precipitation reaction

• Precipitation or immunoprecipitation is the non-covalent interaction between

soluble (small) antigens and soluble antibodies that bind to form an insoluble
precipitate.

• The precipitins need to be bivalent for precipitation.

• Antigens involved need to be bi or polyvalent so that they can bind with multiple
antibodies which aid in the formation of the lattice.

• They can be called precipitinogens.

• These reactions consist of lattice (cross-links) formation when the
corresponding antigen and antibody combine in optimal ratios.
• Lattice formation relies upon the valency of both antibody and
antigen.
• Crosslinked complexes result when bi- or polyvalent antigens interact
with more than one multivalent antibodies.
• If the Ag-Ab complexes formed are too large to stay in solution, visible
precipitation results.
• Excess of either component reduces lattice formation and subsequent
precipitation hence, these should occur at optimal concentrations.
• Antibodies that aggregate soluble antigens are called precipitins.
• Precipitation and agglutination reactions differ in size, solubility of the
antigen and sensitivity.
• Antigens are soluble molecules and larger in size in precipitation
reactions.
• Antigen-Antibody lattice formation is governed by the valency of both
the antibody and antigen:
1. The antibody should be polyvalent (Fab fragments) in order to form
a precipitate.
2. The antigen should be bi- or polyvalent; i.e.. it should possess at
least two copies of an epitope, or have different epitopes that are
capable of reacting with various antibodies in a polyclonal antisera.
• When the ag concentration is very low and that of the ab is relatively
superabundant (zone of ab excess), formation of “small” complexes
occurs.
• If the mixture [ reactants (ag-ab) ] are centrifuged, residual abs will
remain in the supernatant. This area (supernatant) containing excess
antibodies is called PROZONE
• A more antigen is added, large aggregates form, when there is neither
antigen nor antibody in the supernatant, the situation is called
EQUIVALENCE ZONE . This where the maximal precipitation occurs.

• With increasing the amounts of ag , the lattice size becomes too small
to precipitate. This situation is called the POSTZONE (zone of ag
excess). Instead of reaching the plateau, the curve comes back down
to zero