10/19/2022

Antigen Antibody
reactions
WEL COME Dr. Rezina
05/07/20

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Strength of Antigen-antibody interactions

What is antigen and antibody reactions?
Antigen antibody reaction is highly specific
Purpose: • Antibody Affinity- The strength of total noncovalent
interactions between a single antigen binding site on an
In vivo- To eliminate microorganism. antibody and and a single epitope is the affinity of the
In vitro- To detect either antigen or antibody for antibody for that epitope.
diagnostic purpose. • Antibody Avidity- The strength of interactions between a
multivalent antibody and antigen is called the avidity.
• Unknown antigen + Known antibody
• Unknown antibody + Known antigenKnown
antigenP

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Cross-Reactivity
• Although antigen-antibody reaction is highly
specific, in some cases antibody elicited by one
antigen can cross-react with an unrelated
antigen
• Example- Rheumatic heart disease
Serology
Antigen antibody reactions in laboratory
Titre
Is the highest dilution of serum that gives a positive reaction in
many immunological tests
Rising titre- four fold rising is diagnostic

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Use of serological test Use of serological test-condt.

B. diagnosis of Autoimmune diseases
1. Diagnosis of infectious diseases
e.g. Rheumatoid arthritis, SLE
• When the antigen can not be cultured
C. Determination of Blood type and HLA type
e.g. Syphilis and hepatitis A,B, and C
• Cross matching
• When the organism are too dangerous to culture
• Detection of antibody against ABO and Rh
e.g rickettsial diseases
blood group
• When culture techniques are not available.
• Detection of HLA antigens e.g. for tissue
e.g. HIV,EBV
transplantation
• When the organisms takes too long to grow
e.g. Mycoplasma

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Name of serological tests

• Agglutination tests Agglutination
• Precipitation tests
• Enzyme-linked immunosorbent assay( ELISA)
• Chromatographic Immuno assay( CIA) When the antigen is particulate ,the reaction of an
• Dipstick immunoassay
antibody(agglutinin) with the antigen(agglutinogen)
• Complement fixation tests
• Immuno fluorescence test can be detected by clumping of the antigen.
• Radioimmunoassay
• Western blot
• Flow cytometry IgM>IgG more effective as an agglutinating
• Other tests like- capsular swelling test (Quelluntest) Agent

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Types of agglutination tests

Direct agglutination test:
• Direct (active) agglutination tests
Direct agglutination of Antigen with corresponding
Antigen+ Antibody= Clumping Antibody.
e.g.
• Indirect (passive) agglutination tests
Antibody detection by
a) Widal test
The known antibody or antigen is attached to inert
latex particle (latex agglutination test), red cells b) Weil-felix reaction
(haemagglutination test), or protein A of Antigen detection
S.aureus(coagglutination test).
a) Blood grouping
b) Salmonella antigen

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Agglutination test can be performed: Indirect (passive) agglutination:

*On slide-Rapid but less sensitive than tube Specific Antibody or Antigen are passively adsorbed or
chemically coupled to inert particle.
latex, carbon ,RBC etc.
* On tubes - better
e.g.
1.Pregnancy test
* On micro titration plate- Best 2.Widal test-
3.Blood grouping
4. ASO level in serum

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Indirect (passive) agglutination:

A. Latex particle agglutination test

B. Passive hemagglutination test

C.Coagglutination test.

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Indirect (passive) agglutination: Precipitation test

A. Latex particle agglutination test
Pregnancy test
Widal test
B. Passive hemagglutination test
1.Trepenomapallidumhemagglutination test (TPHA)
In precipitation reaction antigen is soluble and the proportion of Antigen &
Antibody must be optimal to give maximum precipitation.
e.g. Elek test
Antibodies that aggregates with soluble antigens are called precipitins
Formation of an Ag-Ab lattice depends on the valency of both the antigen-
antibody
• The antibody must be bivalent; a precipitate will not form with
monovalent Fab fragments

2. Rose-Waaler test (to detect rheumatoid factor)
C.Coagglutination Test.
1. CSF to detect antigens of meningococci,
pneumococci, Haemophilus Influenzae type b.
• The antigen must be either bivalent or polyvalent: that is , it must have at
least two copies of the same epitope, or have different epitopes that react
with different antibodies present in polyclonal antisera.

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Precipitation in agar
Precipitation test
• This is done either single or double diffusion.
• It can also be done in the presence of an
electric field.
Single diffusion- In single diffusion, antibody is
incorporated into agar and antigen is placed into a well. As
the antigen diffuses with time, precipitation rings form
depending on the antigen concentration. The grater the
amount of antigen in the well, the farther the ring will be
from the well. By calibrating the method, such radial
immunodiffusion is used to measure IgG, IgM complement
components and other substances in serum.

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Double diffusion
• Double diffusion-In double diffusion, antigen
and antibody are placed in different wells in
agar and allowed to diffuse and form
gradients. Where optimal proportions occur,
lines of precipitate form. This
method(Ouchterlony) indicates whether
antigens are identical, related but not
identical, or not related.

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Precipitation in agar with an

electric field
• Immunoelectrophoresis- A serum sample is
placed in a well in agar on a glass slide. A
current is passed through the agar, and the
proteins move in the electric field according to
their charge and size. Then a trough is cut into
the agar and filled with antibody. As the
antigen & antibody diffuse towards each other,
they forms a series of arcs of precipitate.

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Counter-immunoelctrophoresis
• This method relies on movement of antigen
toward the cathode and of antibody toward
the anode during the passage of electric
current through agar. The meeting of the
antigen and antibody is greatly accelerated by
this method and is made visible in 30 to 60
minutes. This has been applied to the
detection of bacterial and fungal
polysaccharide antigens in CSF.

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Prozone phenomena

Agglutination may not occur due to Antibody or

antigen excess.

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Differences between agglutination and precipitation

Radioimmunoassay(RIA)
• The method is used for the quantitation of antigen or haptens that can
Agglutination Precipitation
be radioactively labeled.
1. Antigen is particulate 1. Antigen is soluble
• It is based on the competition for specific antibody between the
2. Highly sensitive for antibody 2. Highly sensitive for antigen labeled(Known) and the unlabeled(unknown) concentration of material
detection detection

• The complexes that form between the antigen and antibody can be
3. Agglutination occurs mainly due to 3. Precipitation occurs mainly due to
separated and the amount of radioactivity measured.
antigen(95%) & than to antibody(5%) antibody(95%) & than to antigen(5%)

• The more unlabeled antigen is present, the less radioactivity there is in

the complex.
4. Prozone phenomenon occurs due to 4. Prozone phenomenon occurs due to
high antibody excess high antigen excess

5. Clump consist mainly of antigen. 5.Dense precipitate is formed by optimal

proportion of antigen and antibody.

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Immnunofluoroscenc tests
Principle : Fluorescent dyes can be covalently attached to
antibody and made visible by ultraviolet light in the
fluorescene microscope. Such labeled antibody can be
used to identify antigens.

The Ag-Ab complex are seen fluorescing against a dark back

ground.
Dyes used in fluroscene test
Rhodamine
fluorescein
Acridine

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Immnunofluoroscenc tests

Dye

Anti-antibody

Antibody

Antigen

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Types of Immunofluorescence Test

Uses of Immunofluorescence
Direct FAT: The immunofluorescence reaction is direct
Immunofluroscence test use to detect-
when known labelled antibody interacts directly with unknown ● Bacteria, virus, fungus &parasite
antigen.
● Auto antibodies
Indirect FAT: when two stage process is used.Known antigen is ● Antibody in syphilis
attached to
a slide --- Patients serum is added(unlabelled)--- preparation is ● Leishmanial antibody
washed---- ● Complement components
If patients serum contain antibody against the antigen it will
remain fixed
and can be detected by addition of a fluorescent dye ab to
human IgG and examination by UV microscopy.

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Enzyme linked immunosorbent assay (ELISA) Types of ELISA

Principle: Enzyme system is used to visualize the
specific combination of Antigen with Antibody.
Direct ELISA : Ag detected
Known antibody or antigen is tagged with an Sandwich ELISA : Ag detected
enzyme. If Ag-Ab reaction occur, to detect the Indirect ELISA : Ab detected
presence of enzyme, a substrate is added it will
be catalyzed by the enzyme giving a specific
colour.

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Use of ELISA
• Anti HAV, HBV, HCV, HEV
• HIV
• TORCH

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Complement fixation test

• In this antigen antibody reaction complement
is required.
• The fixation of complement occurs when
antigen combines with specific antibody.
• To find out whether complement is fixed or
not, an indicator called haemolytic system(
sheep red cells+ haemolysin) is used.
• No haemolysis indicates a positive reaction.
• Haemolysis indicates a negative reaction.

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Uses of Complement Complement fixation test

A. Diagnosis of disease- Detection of antibodies.
Examples: Syphilis, (Wassermann reaction –WR)
Hydatid disease, Rickettsial disease, viral disease.

B. Identification of Micro-organism- Detection of

antigenic structure- Rickettsia and viruses.

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Coombs test

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Immunoblotting
Immunoblotting Westernblot: The test is done for the presence
of HIV antibodies in the patients serum. In
• Westernblot
this test HIV proteins are separated
• Southernblot electropho-retically in gel ,resulting in
discrete bands of viral protein. These
proteins are then transferred from the gel
onto a filter paper, and the persons serum is
added.

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• If antibodies are present they bind to the viral
Protein (primarily gp41 and P24 ) and can be
detected by adding antibody to human IgG
labelled with enzyme such Horseradish
peroxidase which produces visible
Color change when substrate is added.
USE: It is done for cofirmatory test for patients
with HIV, or for lyme disease.
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Application of Immunoblot
• i) HIV
• ii) Kala-azar
• iii) Anti HCV
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Southernblot:
This test is used to detect the presence DNA
sequence in DNA samples. DNA fragments
are separated by electrophoresis and
transfer into a filter membrane and detect
the fragment by probe hybridization.
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Polymerase Chain Reaction
It is a primer directed amplification technique by
which very small amount of nucleic acid can be
used to rapidly make millions to billions of
copies of a specific DNA sample within a very
short period of time.
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• The purpose of a PCR (Polymerase Chain
Reaction) is to make a huge number of copies
of a gene. This is necessary to have enough
starting template for sequencing.
• The cycling reactions :
• There are three major steps in a PCR, which are
repeated for 30 or 40 cycles. This is done on an
automated cycler, which can heat and cool the
tubes with the reaction mixture in a very short
time.
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Denaturation at 94°C :
During the denaturation, the double strand
melts open to single stranded DNA, all
enzymatic reactions stop (for example : the
extension from a previous cycle).
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Extension at 72°C :
This is the ideal working temperature for the
polymerase.
The bases (complementary to the template)
are coupled to the primer on the 3' side (the
polymerase adds dNTP's from 5' to 3', reading
the template from 3' to 5' side, bases are
added complementary to the template)
•
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Immunochromatographic test(ICT)
• The membrane-based test device consist of a
chromatography strip, a separator, and an
absorbent pad, all housed in a cassette
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Annealing at 54°C :
The primers are jiggling around, caused by the
Brownian motion. Ionic bonds are constantly
formed and broken between the single stranded
primer and the single stranded template. The more
stable bonds last a little bit longer (primers that fit
exactly) and on that little piece of double stranded
DNA (template and primer), the polymerase can
attach and starts copying the template. Once there
are a few bases built in, the ionic bond is so strong
between the template and the primer, that it does
not break anymore.
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Uses of PCR
• To diagnosis of infectious disease- HIV, HCV, HBV,TB
COVID-19.
• Inherited disorder
• Used in forensic medicine
• HLA typing
• Epidemiologic studies
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Immunochromatographic test(ICT)
or chromatographic immunoassay.
• A rapid and reliable diagnostic test. Most test
results can be read after 5 minutes.A
nitrocellulose membrane- sprayed with
colloidal gold labelled antibody and anti-
human IgG antibody
• A second (assay control) line was sprayed
with colloidal gold labeled IgG & antigen.
• The test is done using test strip ,membrane
card and cassete.
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ICT for H.pylori

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HBV (HBsAg, HBsAb, HBeAg,

HBeAb, HBcAb) rapid test kits

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Uses of ICT Flow cytometry

For diagnosis of -
( Fluorescence – Activated Cell Sorting)
1. Malaria • This test is used to measure the number of
various type of immunologically active blood
2. Filaria
cells.Patients cells are labeled with monoclonal
3. Dengue
antibody that is tagged with fluorescein or
4. Leishmaniasis. rhodamine. Single cells are passed through laser
5. Toxoplasmosis. Beam the number of cell that fluorece is counted
6. Trichomoniasis. by use of a machine called fluorescence –
activated cell sorter. For example, it is used in HIV
infected patients to determine the number of
CD4+ T cells.
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Flow cytometry

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