‫بسم هللا الرحمن الرحيم‬
Faculty of Allied Medical
Sciences
Clinical Immunology & Serology
Practice
(MLIS 201)
ANTIGEN ANTIBODY REACTION
Prof. Dr. Ezzat M Hassan
Prof. of Immunology
Med Res Inst, Alex Univ
E-mail: elgreatlyem@hotmail.com
CLINICAL IMMUNOLOGY & SEROLOGY PRACTICE
(CODE: MLIS 201)
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TEACHING OBJECTIVES:
1. To discuss the factors affecting Ag-Ab reactions
2. To define Affinity & Avidity
3. To know different methods of Ag-Ab reactions
4. To describe the basics and types of precipitation
reactions
6. To describe the basics and types of aggulination
reactions
7. To describe the basics and types of immunoflourescent techniques
8- To describe the basics and types of ELISA
Serology
The science that deals with the properties and reactions of
sera, especially blood serum.
Usually refers to the diagnostic identification of Ag or Ab in
the serum.
Serological assays depends on Antigen-antibody interactions
Nature of antigen-antibody interaction
i.e epitope-paratope binding forces
BINDING OF THE EPITOPE WITH
THE PARATOPE (ANTIGEN
BINDING SITE)
GOOD FIT
Antigen binding site
epitope
POOR FIT
Antigen binding site
epitope
antigen determinant
high attraction
low repulsion
high repulsion
low attraction
Factors Affecting binding of
Ag/Ab
1.Affinity
2.Avidity
3.Law of Mass Action
4.Physical form of Ag
5.Ag:Ab ratio
1-AFFINITY
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Antibody affinity is the strength of the binding
between a single antigenic determinant and a
single combining site on the antibody.
The higher the affinity of the antibody for the
antigen, the more stable will be the interaction.
 2-Avidity
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Avidity is a measure of the overall strength of
binding of multivalent Ag (i.e. antigen with many
antigenic determinants) and multivalent
antibodies.
Reactions between multivalent antigens and
multivalent antibodies are more stable and thus
easier to detect.
2-Avidity (cont.)
The overall strength of binding between a
multivalent Antigen and multivalent
Antibodies
Keq =
104
106
1010
Affinity
Avidity
Avidity
SPECIFICITY
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The ability of an individual Ab binding site to react
with only one antigenic determinant.
The ability of a population of antibody molecules to
react with only one antigen.
Cross Reactivity
• The ability of an individual Ab binding site to react
with more than one antigenic determinant.
• The ability of a population of Ab molecules to react
with more than one Ag
Anti-A
Ab
Ag A
Specific Reaction
Anti-A
Ab
Anti-A
Ab
Ag B
Ag C
Shared epitope
Similar epitope
Cross reactions
Antigen antibody tests
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All tests based on Ag/Ab reactions can be used to
detect either Ag or Ab
Cannot be visualized, thus Detection of
antigen/antibody reactions is difficult
Various laboratory methods have been developed
to make this reaction visible eg. Precipitation,
agglutination, labeled reagents (ELISA, RIA, IF),
…etc.
Methods for detection of Ag-Ab Reactions
1.
Precipitation reactions
(Ag is soluble)
precipitin.
2.
Agglutination reactions
(Ag is particles)
clumping.
3.
Complement fixation reactions.
4.
Labelling methods:
a- Immuno-fluorescence reactions.
b- ELISA.
c- Flow cytometry
METHODS FOR DETECTION OF AG-AB REACTIONS
1- Precipitation reactions
PRECIPITATION
SEROLOGICAL TESTS
PRECIPITATION TECHNIQUES
Precipitation reactions Involve soluble antigens
with antibodies cross-linked and form lattice that
Eventually develops into a visible precipitate.
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In immunodiffusion, antigens and antibodies diffuse through a gel until
they reach the zone of equivalence.
In immunoelectrophoresis, diffusion is combined with electrophoresis.
PRECIPITATION REACTIONS
(a)
Polyclonal Ab can form lattices, or large aggregates with polyvalent
Ag.
(b)
Monoclonal antibody can link only two molecules (epitopes) of antigen
and no precipitate is formed.
( Lattices or
large aggregates )
( no precipitate is formed)
PRECIPITATION CURVE
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The plot of the amount of antibody precipitated versus
increasing antigen concentrations (at constant total Ab)
reveals 3 zones:
1. Zone of antibody excess:
 Precipitation is inhibited.
 Ab not bound to Ag can be detected
in the supernatant;
2. Zone equivalence:
 Maximal precipitation in which
antibody and antigen form large
insoluble complexes.
 No Ab or Ag can be detected in the
supernatant
3. Zone of antigen excess:
 Precipitation is inhibited.
 Ag not bound to Ab can be detected
in the supernatant
PRECIPITATION CURVE
PRECIPITATION TECHNIQUES
1. Tube precipitation test
2. Gel diffusion
 Single radial
 Double
3. Immuno-electrophoresis, immuno-fixation
PRECIPITATION TECHNIQUES
1- Tube precipitation test
 Ag & Ab molecules
diffuse until they reach
equivalence zone
Visible Precipitation
(In gel)
PPT in Fluid
PPT in Gel
PRECIPITATION TECHNIQUES:
2. Gel Immunodiffusion Techniques
 Radial Immunodiffusion
A
single diffusion technique where Ab is put into a
gel and Ag is measured by the size of a precipitin
ring formed when it diffused out in all directions
from a well cut into the gel
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Ouchterlony Double Diffusion
 Both
Ab and Ag diffuse from wells into a gel
medium
RADIAL IMMUNODIFFUSION
A quantitative technique based upon the reaction between an Ag placed
in a well diffuses into an agar containing the Ab.
 During diffusion period the Ag & Ab
continue to react until the zone of
equivalence is reached with
formation of a well-defined ring of
precipitation around the Ag well which
is proportional to the Ag concentration.
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OUCHTERLONY DOUBLE DIFFUSION
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Both Ab and Ag diffuse
independently from
wells into a gel
medium
ELECTROPHORESIS TECHNIQUES
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Electrophoresis separates molecules according
to differences in their electrical charge.
 Rocket
Immunoelectrophoresis
 Countercurrent Immunoelectrophoresis
 Immunoelectrophoresis
 Immunofixation Electrophoresis
3. IMMUNO-ELECTROPHORESIS
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Ag molecultes are separated according to differences
in their electrical charge by electrophoresis
Ab is placed in a trough cut in the agar
Ab diffuse towards Ags
precipitin arcs
Ag
Ag
Ab
Ag
Ab
• Interpretation- Precipitin arc represent individual antigens
MEASUREMENT OF PRECIPITATION
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Turbidimetry
 Passing
light through a cloudy solution of AgAb complexes.
 Net decrease in light intensity correlates to
the concentration of the Ag
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Nephelometry
 Measuring
light scattered at a particular
angle after being passed through a solution
of Ag-Ab complexes.
 Amount of light scattered correlates to the
concentration of the Ag
METHODS FOR DETECTION OF AG-AB REACTIONS
1- Precipitation Reactions
2- Agglutination Reactions
AGGLUTINATION
SEROLOGICAL TESTS
AGGLUTINATION
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Principle
Particulate antigen + antibody –> clumping
 The clumps will be called agglutinates
 Lattice formation (antigen binds with Fab sites of 2
antibodies forming bridges between antigens)
 Agglutination Curve follow
the same roles as in precipitation curve
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evaluation:
Qualitative (y/n),
Semi-quantitative (serum titration)
AGGLUTINATION TESTS
Types of Agglutination Reactions
I.
Direct agglutination Tests
II. Indirect agglutination Tests
III. Agglutination Inhibition Tests
(I) DIRECT AGGLUTINATION TESTS
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Used to determine antibody titer against particulate
antigens (Bacteria, Fungi or RBCs)
Particulate antigen reacts directly with antibodies.
• Applications
– Blood group typing
– Direct Coomb’s Test
– The diagnosis of certain diseases (Bacterial infection
Eg. Syphilis)
–The identification of some viruses.
a) Blood Grouping
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Anti-RBC
RBC
Agglutinate
B) DIRECT COOMBS (ANTIGLOBULIN)TEST
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detects the presence of antibodies on red blood cell
Simply add a second antibody ( Antiglobulin antibody,
Coomb’ Reagent) directed against the immunoglobulin
coating the red cells.
This anti-immunoglobulin antibodies cross link the red
blood cells and result in agglutination.
C) Latex Test
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Ag or Ab are not part of particulate matter but are
attached to it (ANTIBODIES OR ANTIGENS ARE ATTACHED
TO PLASTIC BEADS)
EASY TO DO
Eg. Latex test for RF & CRP
negative
positive
(II) INDIRECT AGGLUTINATION (COOMB’S)TEST
To detect anti- RBC’s antibodies in a serum sample.
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This test is done by incubating RBC’s with the serum sample
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washing out any unbound antibodies
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then
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adding a second anti-immunoglobulin reagent (Coomb’s
Reagent) to cross link the cells resulting in agglutination.
HEMAGGLUTINATION-INHIBITION
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Some viruses (eg. Measles virus) agglutinate RBCs in vitro.
Anti-virus Ab will bind the virus and inhibit
hemagglutination.
APPLICATIONS OF AGGLUTINATION TESTS IN
SEROLOGY
I.
II.
III.
IV.
V.
Determination of blood group antigens.
Diagnosis of cases with Rh incombatability
(erythoblastosis fetalis)
Diagnostics of autoimmune hemolytic diseases
Latex test for detection of Ags or Abs (eg. RF, CRP)
To assess bacterial infections (e.g. Typhoid Fever,
Syphilis)
AGGLUTINATION VS. PRECIPITATION
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Agglutination
Insoluble or particulate Ag or Ab
Ag must have at least two
determinants
Ag xs results in postzone
reaction
Ab xs results in prozone
reactions
Reaction time: minutes to hours
Test results: qualitative or semiquantitative
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Precipitation
Soluble Ag & Ab
Ag must have at least two
determinants
Ag xs results in postzone
reaction
Ab xs results in prozone
reactions
Reaction time: hours to days
Test results: qualitative,
semi-quantitative or
quantitative
METHODS FOR DETECTION OF AG-AB REACTIONS
1- Precipitation Reactions
2- Agglutination Reactions
3- Labelling methods:
a- Immuno-fluorescence
b- ELISA.
c- Flow cytometry
IMMUNOFLUORESCENCE:
METHODS
Direct
Indirect
 Fix specimen on slide
• Fix specimen on slide
 Add Flourescein labeled
• Add antibody specific for
antibody specific for the
desired antigen
 Look for fluorescence
the desired antigen
• Add second Flourescein
labeled antibody
• Look for fluorescence
IMMUNOFLUORESCENCE:
INTERPRETATION
Fluorescence = patient has the antigen
IMMUNOLOGIC LAB TESTS OUTLINE
Agglutination reactions
DAT
IAT
Immunofluorescence
ELISA
ENZYME-LINKED IMMUNOSORBENT ASSAY:
PURPOSE
 Detection of antibodies in patient specimen
 Examples:
• home pregnancy tests
• HIV tests
• tests for some coagulation factors,
cytokines, and autoantibodies
ELISA:
METHOD
 Fix the target (AG or Ab) to wells of microtiter plate
 Add patient specimen to well coated with ligand
 Add AHG with enzyme attached
 Add substrate
 Measure color change
ELISA:
INTERPRETATION
Color change = patient has the antibody
ELISA:
VARIATIONS
 Sandwich immunoassay
• detects antigen (not antibody)
• coat well with antibody
• rest is like ELISA
RADIOIMMUNOASSAY
• detects antibody or antigen
• Label (detector) is a radioactive substance
• otherwise like ELISA or sandwich immunoassay
IMMUNOLOGIC LAB TESTS OUTLINE
Agglutination reactions
DAT
IAT
Immunofluorescence
ELISA
Western blot
WESTERN BLOT:
PURPOSE
 Detection of antibodies in patient specimen
 Most common example: HIV test
WESTERN BLOT:
METHOD
 Make a protein suspension of the target you’re
looking for (e.g., HIV)
 Electrophorese the suspension onto a little gel strip
 Apply the patient’s specimen (containing
antibodies) to the strip
 Add AHG that has an enzyme attached
 Add substrate and look for bands
WESTERN BLOT:
INTERPRETATION
Bands on strip = patient has antibodies to
corresponding proteins
Enough bands = patient is “positive”
IMMUNOLOGIC LAB TESTS OUTLINE
Agglutination reactions
DAT
IAT
Immunofluorescence
ELISA
Western blot
Flow cytometry
FLOW CYTOMETRY:
PURPOSE
 Examples:
• diagnosis of leukemia and
lymphoma
• determination of CD4/CD8 counts in
patients with HIV
IMMUNOLOGIC LAB TESTS OUTLINE
Agglutination reactions
DAT
IAT
Immunofluorescence
ELISA
Western blot
Flow cytometry
STUDY QUESTIONS:
Complete:
The Factors Affecting binding of Ag/Ab are
……………………….
 Compare between: Agglutination &
Precipitation.
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72
‫‪ASSIGNMENT‬‬
‫‪Write on the following subjects:‬‬
‫‪1. Factors affecting Ag-Ab reaction‬‬
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‫أميرة رفعت – اميرة صالح – ايمان محمود – ايمن شكرى – داليا ناصر‬
‫‪2. Precipitation reactions‬‬
‫دعاء عبد هللا – رغدة رشدى – رنا ابراهيم – سمر عبد الحميد – سمية جمال‬
Thanks