The document discusses various aspects of antigen-antibody reactions, including: 1) Antigen-antibody reactions involve the specific binding between epitopes on antigens and paratopes on antibodies. This binding can result in agglutination or precipitation. 2) Reactions are based on non-covalent bonds and involve multiple binding sites on multivalent antibodies binding to epitopes on antigens. 3) Common techniques that rely on antigen-antibody reactions include precipitation, agglutination, immunofluorescence, ELISA, and radioimmunoassay.

1. Antigen and Antibody Reaction
Dr.P.Ruban
Assistant Professor
Department Of Biotechnology
SNMV College of Arts and Science
Coimbatore
Tamilnadu
India

2. Ag-Ab Reaction
 Interaction between Ag-Ab
 It s basis of Humoral and cell mediated
response
 They are very specific
 Binding b/w epitopes and pratopes
 Reaction take place in the surface of the cell
 There is no denature of Ag or Ab during the
reaction
 Entire molecules take part in the reaction
 The reaction form agglutination or precipitation
 The forces acting B/w Ag-Ab are non covalent
forces

3. Salient Features of Ag-Ab Reaction
 Immune complex (Ag+Ab Ag-
Ab Complex)
 Specificity of Ag-Ab reaction (Lock and
Key system)
 Binding sites of Ag- Ab reaction (Epitope
and paratope)
Ag may contain10-50 epitopes may go
up to 200
Ab – are may be Monovalent to
multivalent (5- 10 binding sites)

4.  Binding forces of Ag-Ab (Reaction is due to 3
factores)
--Closeness B/w Ag-Ab
----Intra molecular forces (Non Covalent forces
and Electrostatic forces
Hydrogen, hydrophobic, Vander
walls bonding)
---Affinity of Ab (Strength of bond b/w epitope
and paratope)
 Avinity---Combined strength of multiple bond
intreaction b/wMultivalent Ags and multivalent Ab
 Bonous effect--- The extra strength to the Ag-Ab
complex
 Cross reaction---Reaction of an Ag with a closely

5. Types of Ag-Ab Reaction
 Precipitation
 Agglutination
 Cytolysis
 Complement fixation
 Flocculation
 Opsonization
 Immunofluorescence

6. Precipitation
 This refers to an Ag-Ab reaction b/w a soluble
Ag and its Ab--- resulting – formation of
insoluble precipitation
 The Ab causing precipitation is called precipitin
 Eg..Reaction of Ab with BSA
Mechanism of Precipitation
 Precipitation is due to the formation of Ag-Ab
complex
 The Ag is multivalent and the Ab is bivalent. As
each Ab is a bivalent molecule, it can bridge two
multivalent Ag molecule. This bridging leads to
the formation of a lattice which forms the

7. Precipitation

10. Agglutination
 Agglutination is the clumping of particles. The word
agglutination comes from the Latin agglutinare (glueing
to).
 Agglutination occurs if an antigen is mixed with its
corresponding antibody called isoagglutinin. This term is
commonly used in blood grouping.
 Agglutination is commonly used as a method of
identifying specific antigens
 Because the clumping reaction occurs quickly and is
easy to produce, agglutination is an important technique
in diagnosis.

12. Hemagglutination
 Hemagglutination is the process by which red blood
cells agglutinate, meaning clump or clot. The agglutin
involved in hemagglutination is called hemagglutinin
 In cross-matching, donor red blood cells and recipient's
serum or plasma are incubated together. If agglutination
occurs, this indicates that the donor and recipient blood
types are incompatible.
 Eg: Blood type can be determined by using antibodies that
bind to the A or B blood group antigens in a sample of
blood

13. Immunofluorescence
 Immunofluorescence is a technique used
for light microscopy with a fluorescence microscope and is used
primarily on microbiologicalsamples. This technique uses the
specificity of antibodies to their antigen to
target fluorescent dyes to specific biomolecule targets within a
cell and therefore allows visualisation of the distribution of the
target molecule through the sample.
 Immunofluorescence can be used on tissue sections,
cultured cell lines, or individual cells, and may be used to
analyze the distribution ofproteins, glycans, and small biological
and non-biological molecules
 Immunofluoresence can be used in combination with other, non-
antibody methods of fluorescent staining, for example, use
of DAPI (4',6-diamidino-2-phenylindole) to label DNA.

14. Two classes of immunofluorescence techniques
Primary (or direct) and Secondary (or indirect)
Primary (direct)
 Primary, or direct, immunofluorescence uses a single,
primary antibody, chemically linked to a fluorophore
( fluorescent chemical compound).
 The primary antibody recognizes the target molecule
(antigen) and binds to a specific region called the
epitope.
 The attached fluorophore can be detected via
fluorescent microscopy, which, depending on the
messenger used, will emit a specific wavelength of
light once excited
 The direct attachment of the messenger to the antibody
reduces the number of steps in the procedure, saving

15. Secondary (indirect)
 Secondary, or indirect, immunofluorescence uses two
antibodies; the unlabeled first (primary) antibody specifically
binds the target molecule, and the secondary antibody, which
carries the fluorophore, recognizes the primary antibody and
binds to it.
 Multiple secondary antibodies can bind a single primary
antibody
 This provides signal amplification by increasing the number
of fluorophore molecules per antigen
 This protocol is more complex and time-consuming than the
primary (or direct) protocol above, but allows more flexibility
because a variety of different secondary antibodies and
detection techniques can be used for a given primary antibody

17. ELISA
 ELISA (enzyme-linked immunosorbent assay) is a plate-based
assay technique designed for detecting and quantifying
substances such as peptides, proteins, antibodies and hormones.
 Antigens from the sample are attached to a surface. Then, a
further specific antibody is applied over the surface so it can
bind to the antigen.
 This antibody is linked to an enzyme, and, in the final step, a
substance containing the enzyme's substrate is added.
 The subsequent reaction produces a detectable signal, most
commonly a color change in the substrate.

18.  Performing an ELISA involves at least one antibody with
specificity for a particular antigen. The sample with an unknown
amount of antigen is immobilized on a solid support
 After the antigen is immobilized, the detection antibody is added,
forming a complex with the antigen.
 The detection antibody can be covalently linked to an enzyme, or
can itself be detected by a secondary antibody that is linked to an
enzyme through bioconjugation.
 Between each step, the plate is typically washed with a
mild detergent solution to remove any proteins or antibodies that
are non-specifically bound.
 After the final wash step, the plate is developed by adding an
enzymatic substrate to produce a visible signal, which indicates the
quantity of antigen in the sample.

19. Types Of ELISA

20. Radioimmunoassay
 Radioimmunoassay (RIA) is a very sensitive in
vitro assay technique used to measure
concentrations of antigens (for
example,hormone levels in blood) by use of
antibodies.
 Although the RIA technique is
extremely sensitive and extremely specific,
requiring specialized equipment.
 it remains among the least expensive methods to
perform such measurements. It requires special
precautions and licensing, since radioactive
substances are used.

21. Method
 Classically, to perform a radioimmunoassay, a known
quantity of an antigen is made radioactive, frequently by
labeling it with gamma-radioactive isotopes of iodine, such
as 125-I, attached to tyrosine.
 This radiolabeled antigen is then mixed with a known
amount of antibody for that antigen, and as a result, the two
specifically bind to one another.
 This causes the unlabeled (or "cold") antigen from the serum
to compete with the radiolabeled antigen ("hot") for antibody
binding sites
 As the concentration of "cold" antigen is increased, more of
it binds to the antibody, displacing the radiolabeled variant,
and reducing the ratio of antibody-bound radiolabeled
antigen to free radiolabeled antigen.
 The bound antigens are then separated from the unbound