ANTIGEN - ANTIBODY

REACTIONS
By: Haidy Samir Mohamed khalil
Professor of Medical Microbiology & Immunology
Faculty of Medicine
Helwan University
 Objectives
• Recognize the proerities of Ag-Ab reactions

• Enumerate different types of Ag-Ab reactions

• Explain the principles of different Ag—Ab

reactions

• Identify the uses of different Ag- Ab reactions

 Introduction

•Reactions of antigens and antibodies are highly

specific in the sense that an antibody can react
only with the antigen which induced its
formation.
•These interactions are widely used in vitro for
diagnostic purposes i.e. for the detection and
identification of either antigen or antibody and
are termed serologic reactions.

•In these tests one of the reactants should be

known while the other is unknown.
• The interactions of antigen with antibody may
result in a variety of consequences, including
agglutination if the antigen is particulate,
precipitation if the antigen is soluble, or activation
and fixation of complement.
• The extent of the reaction depends on the
proportions of the interacting antigen and
antibody.
• At high antibody levels, i.e. antibody excess, the
reaction may not occur.
•This is referred to as a "prozone" effect.
Prozone effect
 Types of. Ag-Ab reaction
1- Agglutination
2- Precipitation
3- Complement Fixation
4- Immunoflouresence tests
5- ELIZA
6- Radioimmunoassay
 AGGLUTINATION

•The antigen in the agglutination reaction is in the

form of particles, e.g. suspensions of
microorganisms, cells (e.g. red blood cells), or
latex particles coated with antigens.

•When mixed with the specific antiserum, these

particles become clumped, i.e. agglutinated.
• If one of the reactants (antigen or antibody) is known,
the reaction can be used for the identification of the
other.
• So known antisera are used to identify microorganisms
isolated from clinical specimens.
• On the other hand, known organisms can be used to
detect antibodies in sera of patients.
• There are several types of the agglutination reaction:-
Agglutination:
1-Direct : Slide & tube

2- Coomb’s

3- Latex agglutination

4- Coagglutination

5- Virus haemagglutination inhibition test

6- Heterophil antibody agglutination test

 1. Direct Agglutination:
can be done in:

A- slide
B- Tube
 A- The slide method

• It is rapid and useful in identification and typing of

isolated organisms by mixing a drop of the bacterial
suspension with known antisera.
• Clumping occurs if the serum is specific to the
organism under test.
• The slide method is also used in blood grouping.
 B- The tube method

• it is a quantitative test, which is used to determine the

amount of antibodies in the serum of patients.
• It is done by mixing serial dilutions of the patient's
serum (1/10, 1/20, 1/40, 1/80, etc.) with a constant
amount of a known bacterial suspension, suspected to
be the cause of the disease.
• The last tube showing visible agglutination will reflect
the serum antibody titer of the patient.
•The classical application of the tube method is
the Widal test used to detect antibodies to
salmonella in the serum of patients suspected to
have enteric fever.
•The agglutination tests are also used in diagnosis
of brucellosis, and other diseases.
• In diagnosis of such infectious diseases, two serum
samples separated by 7-10 days interval should be
tested.

• A rising antibody titer of two folds or more is

diagnostic.
• The titer in this test is 1/160 = Highest dilution that shows agglutination
• The titer in this test is 1/40 = Highest dilution that shows agglutination
2. Antiglobulin Agglutination test = Coomb's test:
•This test is used to determine the presence of Rh
incompatibility which causes "erythroblastosis
foetalis".
•Most anti-Rh antibodies are incomplete IgG
(unagglutinable) antibodies which can only coat
the Rh positive RBCs, but can not bridge
between two RBCs to cause agglutination.

•These antibodies can be detected by the

antiglobulin Coomb's test which is performed in
two ways:
• Indirect Coomb's test:
• The mother's serum -containing incomplete anti-Rh
antibodies- is mixed with Rh-positive RBCs (group O).
After incubation, the mixture is centrifuged.
• The deposit containing red cells coated with incomplete
antibodies is washed, and antihuman globulin is added,
and the tubes incubated.
• The antihuman globulin causes agglutination by linking
together the incomplete antibody molecules.
• Direct Coomb's test:
• This test can detect incomplete Rh antibodies coating
the RBCs of the newborn in erythroblastosis foetalis.
• The antihuman globulin is added directly to a washed
suspension of the newborn RBCs, agglutination
occurs.
• Both the direct and indirect Coomb's tests are also
used to detect incomplete antibodies in autoimmune
haemolytic anaemias.
3. Latex or Passive Agglutination:
• It is an agglutination reaction in which inert particles,
e.g. latex or RBCs are coated with various antigens or
antibodies.
• These particles are aggregated in the presence of
specific antibody or antigen, respectively.
• Examples of passive agglutination include the
following:-
a-Immunologic pregnancy test:
• It depends on the appearance of human chorionic
gonadotrophic hormones (HCG) in the urine of
pregnant females.
• The test is done by adding a drop of latex particles
coated with anti-HCG to a drop of urine; agglutination
occurs if HCG is present in urine.
b- Rheumatoid factor
c-C-reactive protein (CRP)
d-anti-streptolysin 'O' (ASO) tests, used for diagnosis of
acute rheumatic fever
 4- Co-agglutination

- Staphylococcus aureus (Cowan I strain) has

specific receptors for the antibody ( attached
from fc portion, leaving the fab fragment free to
bind to the antigen.
- The same idea of the latex
5. Haemagglutination Inhibition assays.
•They are used to determine whether an
individual has been exposed to certain types
of viruses that cause agglutination of red
blood cells.
•The patient serum containing specific antiviral
antibodies is mixed with a known virus, then
RBCs are added, the antibodies will bind to the
virus and interfere with haemagglutination of
RBCs by the virus i.e. virus haemagglutination
inhibition.
6- Heterophil antibodies agglutination tests
1- Paul Bunnell test Diagnose infectious mononucleosis
(Epstein Barr) Virus
II- Cold agglutinins
Patients with mycoplasma pneumonia
III -VDRL and RPR test: In sera of syphilitic patients
 Types of. Ag-Ab reaction
1- Agglutination
2- Precipitation
3- Complement Fixation
4- Immunoflouresence tests
5- ELIZA
6- Radioimmunoassay
 PRECIPITATION
•This is an antigen antibody reaction in which the
antigen is soluble.
•Precipitation reactions can be done in solution
or in agar gel
A- Precipitation in solution:
•This reaction can be made quantitative
•It is used primarily in research.
B- Precipitation in agar:
•In this technique, diffusion of antigen and
antibody is allowed to occur in agar gel.

•It can also be done in presence of an electric

field.
1- Double diffusion (Ouchterlony):
•The antigen and antibody are placed in different
wells punched in the agar.
•Both will diffuse in the agar and where they
meet at optimal proportions, precipitation bands
will appear.
•This method can show whether antigens are
identical, related but not identical, or not
related.
•A clinical application for this method is the Elek's
test which is used to prove the toxigenicity of
diphtheria bacilli

•A strip of filter paper, saturated with diphtheria

antitoxin, is embedded in serum agar.
•A heavy inoculum of the diphtheria bacillus to
be tested is inoculated at right angle to the
strip of filter paper.

• Plates are examined after 2 days incubation

at 37°C.
•If the organism is toxigenic, the toxin will
diffuse sideways from the inoculum and the
antitoxin diffuses from the filter paper, and
where they meet at optimal concentrations, a
precipitate is formed which will appear as
fine white lines.
2- Single diffusion:
•The antibody is incorporated in the agar before
pouring it in the plates.
•The antigen is placed in a well punched in this
agar.
•The antigen diffuses in all directions, and where
its concentration is optimal in relation to the
antibody, a precipitation ring will form around
the well.
•The diameter of the ring depends on the antigen
concentration.

•This technique is used to estimate the quantity of

the various immunoglobulin classes, complement
components and other substances in human
serum.
3- Precipitation in agar with an electric field:
•Immunoelectrophoresis: No details
•Western Blots (immunoblots): No Details
 Types of. Ag-Ab reaction
1- Agglutination
2- Precipitation
3- Complement Fixation
4- Immunoflouresence tests
5- ELIZA
6- Radioimmunoassay
 COMPLEMENT FIXATION (CF)

•This is an antigen antibody reaction that occurs

in the presence of a third component known as
the complement.

•The antigen unites with its specific antibody and

the resulting complex fixes the complement.
•The CF test is used for diagnosis of many
diseases by detecting complement fixing
antibodies in the serum of patients, as in
syphilis, whooping cough, chronic gonorrhoea,
typhus, small pox and other diseases.
•N.B. This test is infrequently used nowadays and
is replaced by more sensitive and faster tests.
 Types of. Ag-Ab reaction
1- Agglutination
2- Precipitation
3- Complement Fixation
4- Immunoflouresence tests
5- ELIZA
6- Radioimmunoassay
 IMMUNOFLUORESCENCE

•These are antigen antibody reactions in which we

use fluorescein labelled antibodies.
•Fluorescein is a dye which emits greenish
fluorescence under ultraviolet light (UV) by the
fluorescent microscope , and it can be tagged to
immunoglobulin molecules.
•There are two ways for doing the test:
1- Direct Immunofluorescence:
• In this test, fluorescein labelled antibodies are layered over the
antigen fixed on a slide.
• They are left to react for some time then the excess unattached
antibody is washed thoroughly.
• The slide is then examined by the fluorescent microscope
under ultraviolet light.
• The site where the antibody adheres to its specific antigen will
be seen as apple green fluorescence. T
• his method can be used to detect bacteria, viruses and antigens
in tissues or in pathological samples.
2- Indirect Immunofluorescence:
•The test is used to detect antibodies in serum of
patients.
•The serum is layered on the antigen preparation
which is fixed on a slide; they are allowed to
react for some time, then the excess is washed.
• Whether there is antibody in the serum, that adhered
to the antigen, or not is determined by adding a
fluorescein labelled anti-globulin which will attach to
the antibody if present in patient's serum and give
positive fluorescence under UV light.
• This method is used in the serologic diagnosis of
syphilis (caused by Treponema pallidum) to detect
anti-treponemal antibodies. This is known as
Fluorescent treponemal antibody (FTA) test.
 Types of. Ag-Ab reaction
1- Agglutination
2- Precipitation
3- Complement Fixation
4- Immunoflouresence tests
5- ELIZA
6- Radioimmunoassay
 ENZYME-LINKED IMMUNO SORBENT ASSAY
(ELISA)

•This technique is very sensitive and does not

require specialized equipment and avoids the
hazards of radioactivity.

•The method depends on conjugation of an enzyme

to either antigen or antibody, then the enzyme
activity on a substrate is used as a quantitative
measure.
•To measure antibody, the indirect method is
used.
•A known antigen is fixed to a solid phase (e.g.
plastic cup or microplate), incubated with the
test serum, then washed to remove excess
unattached antibody and re-incubated with
antiglobulin labelled with a suitable enzyme
(e.g. horseradish peroxidase).
•The labelled antiglobulin will attach to the
antibody bound to the fixed antigen.

• After washing, the enzyme activity is measured

by adding a specific substrate and measuring the
degree of colour change.
• To measure an antigen, the double antibody technique
is used.
• A known antibody is fixed to the solid phase.
• The test material containing antigen is added and the
excess washed.
• A specific known antibody labelled with enzyme is
added.
• After washing, a substrate is added and the enzyme
activity measured colourimetrically and related to
antigen concentration.
 Types of. Ag-Ab reaction
1- Agglutination
2- Precipitation
3- Complement Fixation
4- Immunoflouresence tests
5- ELIZA
6- Radioimmunoassay
 RADIOIMMUNO ASSAY (RIA)
• This is a sensitive method used to measure antigens or
antibodies that can be radioactively labelled.
• There are many variations of the test.
• However, the solid phase RIA is a popular method.
• The principle of the assay method is that radio-iodine
(125I) labelled antigen (e.g. thyroid hormones T3 or T4,
hepatitis B antigen) competes with a non-labelled
antigen in a test sample, for a fixed amount of a specific
antibody in a limited time.
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• The test is done by adding the serum sample (test
antigen) to antibody adsorbed to solid phase (tubes or
beads), then the labelled antigen is added and
incubated for a limited time.

• After decanting the supernatant, the remaining bound

radioactivity is measured in a gamma counter and the
percentage bound labelled antigen is calculated.
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•The concentration of test antigen is deduced
from a standard curve drawn between
concentrations of known standards (run at the
same time with the test) and the percentage
bound radiolabeled antigen.
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• N.B. The shelf life time of the reagents used in RIA is 2 weeks to 2
months as compared to that of the reagents used in the ELISA which
is 6-12 months. In addition, the RIA reagents are hazardous while
ELISA reagents are not.
 Types of. Ag-Ab reaction
1- Agglutination
2- Precipitation
3- Complement Fixation
4- Immunoflouresence tests
5- ELIZA
6- Radioimmunoassay
 FLOW CYTOMETRY

• Flow cytometers are instruments capable of analyzing

properties of single cells, in a fluid sheath, as they pass
through an orifice at high velocity (50,000 cells/minute).
• They can sort out and count cells with certain
characteristics from the general population.
• Properties measured include physical characters such as
size, volume, refractive index, viscosity and chemical
features such as content of DNA or RNA, proteins and
enzymes.
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•These properties are detected by measuring
light scatter, cell volume and fluorescence after
staining the cells with fluorescein labelled
monoclonal antibodies to any specific cell
surface markers.
 Personal contact data
• Haidy.khali@med.helwan.edu.eg
• Haidymicrobiology@yahoo.com

• 01091584654

• Haidy Khalil