ANTIGEN-ANTIBODY

REACTIONS
 Introduction
✓ Antigens and antibodies binds to each other specifically in
an observable manner. The interaction between the Antigen
and Antibody is known as Antigen-Antibody Reactions.
✓ It may be abbreviated as Ag-Ab reaction.

Purposes of Antigen and Antibodies Reactions:

✓ Body- they form the basis of Antibody-mediated immunity
in infectious diseases (hypersensitivity or autoimmune
diseases).
✓ Laboratory- help in the diagnosis of reactions.
✓ Epidemiological Surveys- assist in the identification of
infectious agents and non-infectious agents.
✓ Serological Reactions- Ag-Ab reaction in vitro.
 Stages of Ag-Ab Reactions
✓ Primary Stage- the reaction involves formation of Ag-Ab
Complex.
✓ Secondary Stage- leads to observable events like
precipitation, agglutination etc.
✓ Tertiary Stage- includes destruction of Ag or its
neutralization.
 Features of Ag-Ab Reactions
1) The reaction is specific, i.e. an Ag binds with its specific
homologous Ab and vice-versa.
2) Entire molecule is involved in the reaction (not fragments).
3) There is no denaturation of the Ag or the Ab during the
reaction.
4) The combination occurs at the surface.
5) The combination is firm but reversible.
6) Ag and Ab can combine in varying portions.

✓ Affinity- refers to the intensity of attraction between the Ag

and Ab molecules.
✓ Avidity- is the strength of the bond after the formation of the
Ag-Ab Complex.
 Measurement of Ag and Ab
✓ It is defined in terms of Mass (Eg: mg nitrogen) are more
commonly as units of titre.
✓ The Ab-titre of a serum is the highest dilution.

The important parameters of serological tests are:

a) Sensitivity- refers to the ability of the test to detect even
very minute quantities of Ag or Ab.
b) Specificity- refers to the ability of the test to detect
reactions between homologous Ag and Ab.
 Serological Reactions
✓ The types of Ag-Ab Reactions are:
1) Precipitation Reaction
2) Agglutination Reaction
3) Complement Fixation
4) ELISA (Enzyme Linked ImmunoSorbent Assay)
5) Immunofluroscence
 Precipitation Reaction
✓ When a soluble Ag combines with its Ab in the presence
electrolyte (NaCl) at a suitable temp and pH.
✓ It forms an insoluble precipitate of Ag-Ab Complex.
✓ The Ab causing precipitation is called precipitin and the
reaction is called precipitation reaction.
 Precipitation Curve
Precipitation Curve shows three zones:
✓ Zone of Ab axis
✓ Zone of Equivalence
✓ Zone of Ag axis
 Radial Immunodiffusion
✓ In these methods agar gel or similar gels are used on plates
or petriplates.
✓ Both Ag and Ab diffuse freely in the gel system in all
directions.
✓ At a certain point depending on the rate of diffusion and
concentration of the reactants, a zone of equivalence will be
formed (seen as visible precipitation).
✓ It is of two types:
a) Single Radial Immunodiffusion- Ab is put into a gel and
Ag is put in a well cut into the gel and a precipitin ring
formed when Ag diffuses out in all directions.
b) Double Radial Immunodiffusion- Both Ab and Ag diffuse
from wells into a gel medium.
Single vs. Double Immunodiffusion
 Agglutination
✓ When a particular Ag is mixed with its Ab’s in the presence of
electrolytes at suitable temp and pH, the particles are
clumped or agglutinated.
✓ The Ab of the serum causes the cellular Ag’s to form clumps
and these are called Agglutinins.
✓ The particulate Ag that are aggregated are termed as
Agglutinogens.
 Slide Agglutination
✓ This is a rapid method to determine the presence of
agglutinating Abs.
✓ Eg: The test is used for blood grouping (haemagglutination)
and cross matching. If granulation occurs the test is positive.
 Tube Agglutination
✓ It is a standard method for quantitate estimation of Ab.
✓ The serum containing Ab is diluted serially with saline in
several small test tubes, to which a constant volume of Ag
suspension is added.
✓ The tubes are incubated until visible agglutination is
observed.
✓ Eg: Serological diagnosis of typhoid, brucellosis and typhus
fever. Widal test is used for typhoid fever.
Tube Agglutination
 Passive Agglutination Test
✓ Similar haemagglutination test, but the physical nature of the
reaction is altered,
✓ The Ag is coated on the surface of a carrier particle and
thereby helps to covert a precipitation reaction into an
agglutination reaction.
✓ The carrier particle used can be RBC latex particles or
bentonite.
✓ Eg: Diagnosis of Rheumatoid Arthritis.
Passive Agglutination Test
 Neutralization
✓ The binding of Ab to microbial epitopes or soluble molecules
(e.g. toxins) which inhibits their binding to host cells.
✓ Abs are mostly IgG & IgA.
✓ Used to identify toxins and viruses.
 Opsonisation
✓ Opsonization is a term that refers to an immune process
where particles such as bacteria are targeted for destruction
by an immune cell known as a phagocyte .
✓ The process of opsonization is a means of identifying the
invading particle to the phagocyte.
✓ The process includes, a pathogen is marked (tagged) for
ingestion and destruction by phagocytic cells.
 Complement Fixation
✓ Lysis of RBC or bacteria requires some non-specific
unstable components of fresh serum which are called
complement.
✓ The complement system compromises of 11 proteins and
are present in every individual.
✓ They bind to Fc component of Ab involved in Ag-Ab
Complex.
✓ If Ab specific for the Ag is present in the serum, Ag-Ab
complex will be formed that will fix the complement.
Complement Fixation
 Radioimmunoassay (RIA)
✓ Radioimmunoassay (RIA) is an in vitro assay that measures
the presence of an antigen with very high sensitivity.
✓ Basically any biological substance for which a specific
antibody exists can be measured, even in minute
concentrations.
✓ The technique was introduced in 1960 by Berson and Yalow
as an assay for the concentration of insulin in plasma.
 Principle of RIA
✓ RIA involves the separation of a protein (from a mixture)
using the specificity of antibody-antigen binding and
quantification using radioactivity.
✓ RIA utilize a radioactive label (usually 125I, 3H or 14C), which
emits radiation that can be measured with a beta or gamma
counter.
Steps of RIA
 ELISA- Enzyme Linked ImmunoSorbent
Assay
✓ In 1971, enzyme labelled Ag’s and Ab’s were developed as
serological reagents for the assay of Ab’s and Ag’s .
✓ Used for qualitative and quantitative analysis.
✓ The ligand used here is a molecule which can detect the Ab
and is covalently coupled to an enzyme such as peroxidase,
betagalactosidase, alkaline phosphatase etc.
✓ Depend on enzyme conjugated to Ab reacting with a specific
substrate to produce a color reaction.
 Principle of ELISA

✓ Principle is based on the formation of Ag-Ab complex, which

is detected by chromogenic detection using enzyme
conjugated secondary Ab.
✓ The conjugated enzyme acts on a specific substrate called
chromogenic substrate, and generates a colored reaction
product.
✓ This product is qualitatively and quantitatively read using an
ELISA plate reader.
Steps of ELISA
 ELISA Equipment- Microtitrewell
✓ Made up of polystyrene.
ELISA Equipment-Micropipette
ELISA Equipment- Washer
ELISA Equipment- Reader
 Types of ELISA
✓ There are 3 types:
✓ Indirect ELISA- This technique is used for the detection of HIV. The
envelope proteins are developed by recombinant technology and coated
on the surface of the microtitre plates. Suspects serum are added, and
unbound proteins are washed off.
✓ Sandwich ELISA- Used to detect the presence of Ag in the sample. The
well is coated with Ab specific to the Ag and then suspect serum is added
and allowed to react. The wells are washed off to remove the unbound
Ag’s.
✓ Competitive ELISA- Ab coated microwell.
✓ Serum Ag and labeled Ag added together (competes for binding to the
Ab).
✓ Ab-Ag enzyme complex bound is inversely related to the conc. of Ag
present in the sample.
✓ Increased serum Ag results in reduced binding of Ag-enzyme conjugate
with the antibody producing less enzyme activity and (yellow) color
formation.
✓ Used to determine small molecules like T3, T4 and Progesterone.
Types of ELISA
 Immunofluorescence
✓ Fluorescence is the property of absorbing light rays of one particular
wavelength and emitting rays with a different wavelength.
✓ Fluorescent dyes show up brightly under UV light as they convert into
visible light.
✓ Coons et al (1942) showed that labeled dyes can be conjugated to Ab’s
and these labeled Ab’s can be used to detect Ag’s.
✓ Eg: Fluorescein, Phycoerithrin.
 Applications of Ag-Ab Reactions
1) Determination of blood groups for transfusion.
2) Serological ascertainment of exposure to infectious agents.
3) Development of immunoassays for the quantification of various
substances.
4) To detect the presence or absence of protein in serum.
5) Determining the characteristics of certain immunodeficiency disease.