Antigen-Antibody Reactions
2011
•Features
•Examples
in vivo
in vitro
John F Kearney
410 Shelby Building
4-6557
jfk@uab.edu
Learning Objectives
• To understand
– The features of the antigen-antibody interaction
• Reversible
• Affinity
• Avidity
– What makes a good antigen
– Examples of in vivo antigen-antibody interactions
– Examples of in vitro antigen-antibody interactions
Structure of an IgG Antibody
Features of the Antigen-Antibody
Interaction
A reversible bimolecular association
involving various non-covalent interactions
Is similar to an enzyme-substrate interactions,
but not lead to an irreversible chemical alteration
However catalytic antibodies have been
described
Non-covalent binding
Nature of Ag/Ab Reactions
- Four types of non-covalent forces operates over a very short distance
( generally 1 angstrom )
Specificity
• The ability of an individual antibody combining site
to react with only one antigenic determinant.
Cross Reactivity
• The ability of an individual Ab combining site
to react with more than one antigenic
determinant.
• The ability of a population of Ab molecules to
react with more than one Ag
Cross reactions
Anti-A
Ab
Anti-A
Ab
Anti-A
Ab
Ag A
Ag B
Ag C
Shared epitope
Similar epitope
K.R. Deepthi
Affinity
• Strength of the reaction between a single antigenic
determinant and a single Ab combining site
High Affinity
Low Affinity
Ab
Ab
Ag
Ag
Affinity =  attractive and repulsive forces
Calculation of Affinity
Ag + Ab  Ag-Ab
Applying the Law of Mass Action:
Keq =
[Ag-Ab]
[Ag] x [Ab]
Avidity
• The overall strength of binding between an Ag
with many determinants and multivalent Abs
Keq =
104
Affinity
106
Avidity
1010
Avidity
Factors Affecting Measurement of
Ag/Ab Reactions
• Affinity
• Avidity
Ab excess
Ag excess
• Ag:Ab ratio
• Physical form of Ag
Equivalence – Lattice formation
Precipitation Reactions
( no precipitate is formed
( Lattices or
if an Ag contains only a
large aggregates ) single copy of each epitope )
FIGURE 6-4
Precipitation reactions in fluids yield a precipitin curve.
Antigen-Antibody Binding
Epitope
Features of the Antigen-Antibody
Interaction
•Reversible
Non-covalent Interactions
•Affinity
Measure of the strength of the binding
Ease of association or dissociation
•Avidity
Increase in affinity due to
multivalent binding
The summation of multiple affinities
Antigen-Antibody Binding
Terminology
Antigen
Original - Substance able to generate antibody
More general - Substance that can be recognized
by antibody or T cells
Immunogen Substance able to generate antibody
or T cell responses
What Makes a Good Antigen?
Parameter
Size
Chemical
Composition
Similarity to selfantigens
Dosage and route
of administration
Use of adjuvants
Genetic
composition of
recipient
Immunogenicity
Large > Small
Proteins>Carbohydrates>Lipids
Multiple differences > Few
differences
Subcutaneous > Oral
Increase titer and affinity
Major Histocompatibility
Complex (HLA)
For T cell responses antigens should be degradable
Terminology
Antigen
Original - Substance able to generate antibody
More general – able to bind antibody
Immunogen Substance able to generate antibody or T cell
responses
Hapten
Non-immunogenic substance
Usually low molecular weight
Can induce antibody formation when coupled
to a larger “carrier” molecule
Hapten, DNP
Protein Carrier
Bovine Serum Albumin
Immunize with:
DNP
BSA
DNP-BSA
Antibodies formed:
None
Anti-BSA
Anti-DNP
Anti-BSA
Anti-DNP-BSA
Examples of Haptens
Cortisol
Thyroid hormones
Estradiol
Testosterone
Penicillin
Antigen-Antibody Interactions
in vivo
Direct Inhibition
Toxin
© 1998 Gold Standard Multimedia Inc.
Inhibit attachment of bacteria to cells
Pseudo colored EM image of Helicobacter
pylori attached to a gastric epithelial cell
Would antibodies be effective at
preventing H. pylori attachment?
1. Yes
2. No
o
0%
N
Ye
s
0%
Antibodies Enhance
Phagocytosis
Opsonization
Fc Receptor
© 1998 Gold Standard Multimedia Inc.
Complement Lysis
Complement activation can be initiated by antigen-antibody complexes
© 1998 Gold Standard Multimedia Inc.
Antigen-Antibody Reactions In Vivo
The Good
•Neutralization of viruses and toxins
•Opsonization of pathogens
•Complement lysis of bacteria
•Prevention of bacterial adherence
The Bad
•Autoimmunity - myasthenia gravis
•Transfusion reactions
The Ugly
•Allergy
Antigen-Antibody Interactions
in vitro
Techniques
Hemagglutination
Used to identify blood group antigens
or antibodies to them
Agglutination/Hemagglutination
• Definition - tests that have as their endpoint
the agglutination of a particulate antigen
– Agglutinin/hemagglutinin
• Qualitative agglutination test
– Ag or Ab
+

Agglutination Reactions
-visible clumping by interaction between Ab & a particulate antigen such as RBC,
latex beads.
-routinely performed to type RBCs for blood transfusion.
+
+
+
(control)
FIGURE 6-7
Demonstration of hemagglutination using Ab against sheep
red blood cells (SRBCs).
Hemagglutination
Used to identify blood group antigens
or antibodies to them
ELISA
Enzyme Linked Immunosorbent Assay
HIV Testing
HIV Ag
Human
Serum
Colorless →
Enz-Goat
Anti-Human IgG Yellow
1. ELISA, determine if patient sera is + or – for
antibodies to HIV
2. Western Blot, determine which viral antigens the patient has made
antibodies to.
Immunofluorescence
Fluorochromes
-Fluorescein (490→517nm)
-Rhodamine (515→546nm)
-Phycoerythrin
: absorb light of one wavelength & emit
fluorescence at a longer wavelength than
fluorescein.
FIGURE 6-14
mIgM-producing B cells indirectly stained with rhodamine-conjurated
secondary Ab under a fluorescence microscope.
Bacillus anthracis B. thuringiensis
B. subtilis
Examples of markers to dissect heterogeneity of immune cells
Optical Design
PMT 5
PMT 4
Sample
PMT 3
Flow cell
Dichroic
Filters
Scatter
Sensor
PMT 2
PMT 1
Laser
Bandpass
Filters
Hematoxlin eosin staining
Staining of spleen with antibodies to distinguish cell types
Marginal zone macrophages
Metallophilic macrophages
IgM +B cells
CD4/CD8 T ells
Pregnancy Testing
Assay for human chorionic gonadotrophin
1.
Bioassay (Rabbit Test)
2.
Agglutination inhibition
Summary of Antigen-Antibody
Interactions
Specificity depends on the variable
regions of the immunoglobulin heavy and
light chains
In vivo
• After antibody binds antigen, the outcome
depends on the constant region of the
heavy chain
• Effector functions are determined by
antibody isotype
Summary of Antigen-Antibody
Interactions
In vitro
• All assays depend on exquisite specificity
of the antibody
• Assays differ in the means used to detect
the bound antibody or bound antigen
• Sensitivity: For example, ELISA and
Western blot>>Agglutination
Affinity:
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is due to covalent bonds
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avidity.
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imported automobile.
is a measure of the
strength of antigenantibody binding.
Answers 2 and 4 are
correct.
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1.
Pregnancy Testing
Assay for human chorionic gonadotrophin
1.
Bioassay (Rabbit Test)
2.
Agglutination inhibition
3.
ELISA
Evolution of Flow Instruments
WA Bonner, HR Hulett, RG Sweet and LA Herzenberg,
Fluorescence Activated Cell Sorting, Review of Scientific Instruments 43, 1972
Slide Kindly Supplied by Compucyte