antigen-antibody reactions 2011 features examples in vivo in vitro john f kearney 410 shelby...
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Antigen-Antibody Reactions 2011
• Features
• Examples in vivo in vitro
John F Kearney410 Shelby Building
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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
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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
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Non-covalent binding
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- Four types of non-covalent forces operates over a very short distance
( generally 1 angstrom )
Nature of Ag/Ab Reactions
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Specificity• The ability of an individual antibody combining site to
react with only one antigenic determinant.
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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
Anti-A Ab
Ag A
Anti-A Ab
Ag B
Shared epitope
Anti-A Ab
Ag C
Similar epitope
Cross reactions
K.R. Deepthi
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Affinity = attractive and repulsive forces
Ab
Ag
High Affinity
Ab
Ag
Low Affinity
Affinity
• Strength of the reaction between a single antigenic determinant and a single Ab combining site
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Calculation of Affinity
Ag + Ab Ag-Ab
Keq = [Ag-Ab]
[Ag] x [Ab]
Applying the Law of Mass Action:
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Avidity• The overall strength of binding between an Ag
with many determinants and multivalent Abs
Keq = 104
Affinity106
Avidity1010
Avidity
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Factors Affecting Measurement of Ag/Ab Reactions
• Affinity
• Avidity
• Ag:Ab ratio
• Physical form of Ag
Ab excess Ag excess
Equivalence – Lattice formation
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Precipitation reactions in fluids yield a precipitin curve. FIGURE 6-4
( Lattices or large aggregates )
( no precipitate is formed if an Ag contains only a single copy of each epitope )
Precipitation Reactions
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Antigen-Antibody Binding
Epitope
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Features of the Antigen-Antibody Interaction
• ReversibleNon-covalent Interactions
• AffinityMeasure of the strength of the bindingEase of association or dissociation
• AvidityIncrease in affinity due to
multivalent bindingThe summation of multiple affinities
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Antigen-Antibody Binding
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Terminology
Antigen Original - Substance able to generate antibodyMore general - Substance that can be recognizedby antibody or T cells
Immunogen Substance able to generate antibodyor T cell responses
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What Makes a Good Antigen?
Parameter ImmunogenicitySize Large > SmallChemicalComposition
Proteins>Carbohydrates>Lipids
Similarity to self-antigens
Multiple differences > Fewdifferences
Dosage and routeof administration
Subcutaneous > Oral
Use of adjuvants Increase titer and affinityGeneticcomposition ofrecipient
Major HistocompatibilityComplex (HLA)
For T cell responses antigens should be degradable
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Terminology
Antigen Original - Substance able to generate antibodyMore general – able to bind antibody
Immunogen Substance able to generate antibody or T cellresponses
Hapten Non-immunogenic substanceUsually low molecular weightCan induce antibody formation when coupled to a larger “carrier” molecule
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Protein CarrierBovine Serum Albumin
Immunize with: Antibodies formed:DNP NoneBSA Anti-BSA
DNP-BSA Anti-DNP Anti-BSA Anti-DNP-BSA
Hapten, DNP
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Examples of Haptens
CortisolEstradiol Testosterone
Thyroid hormonesPenicillin
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Antigen-Antibody Interactionsin vivo
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Direct Inhibition
© 1998 Gold Standard Multimedia Inc.
Toxin
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Inhibit attachment of bacteria to cells
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Pseudo colored EM image of Helicobacter pylori attached to a gastric epithelial cell
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Would antibodies be effective at preventing H. pylori attachment?
Yes N
o
0%0%
1. Yes
2. No
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Antibodies Enhance Phagocytosis
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Opsonization
© 1998 Gold Standard Multimedia Inc.
Fc Receptor
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Complement Lysis
Complement activation can be initiated by antigen-antibody complexes
© 1998 Gold Standard Multimedia Inc.
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Antigen-Antibody Reactions In Vivo
• 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
The Good
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Antigen-Antibody Interactionsin vitro
Techniques
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Hemagglutination
Used to identify blood group antigens or antibodies to them
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Agglutination/Hemagglutination
• Definition - tests that have as their endpoint the agglutination of a particulate antigen– Agglutinin/hemagglutinin
+
• Qualitative agglutination test– Ag or Ab
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FIGURE 6-7
Demonstration of hemagglutination using Ab against sheep red blood cells (SRBCs).
Agglutination Reactions
+ + + (control)
- visible clumping by interaction between Ab & a particulate antigen such as RBC, latex beads.-routinely performed to type RBCs for blood transfusion.
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Hemagglutination
Used to identify blood group antigens or antibodies to them
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ELISAEnzyme Linked Immunosorbent Assay
HIV Ag HumanSerum
Enz-GoatAnti-Human IgG
Colorless →Yellow
HIV Testing
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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.
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Immunofluorescence
mIgM-producing B cells indirectly stained with rhodamine-conjurated secondary Ab under a fluorescence microscope.
FIGURE 6-14
Fluorochromes- Fluorescein (490→517nm)- Rhodamine (515→546nm)- Phycoerythrin : absorb light of one wavelength & emit fluorescence at a longer wavelength than fluorescein.
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Bacillus anthracis B. thuringiensis B. subtilis
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Examples of markers to dissect heterogeneity of immune cells
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Optical Design
PMT 1
PMT 2
PMT 5
PMT 4
DichroicFilters
BandpassFilters
Laser
Flow cell
PMT 3
Scatter
Sensor
Sample
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Hematoxlin eosin staining
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Staining of spleen with antibodies to distinguish cell types
Marginal zone macrophages
Metallophilic macrophages
IgM +B cells
CD4/CD8 T ells
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Pregnancy Testing
1. Bioassay (Rabbit Test)
2. Agglutination inhibition
Assay for human chorionic gonadotrophin
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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
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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
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Affinity:
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1. is due to covalent bonds between antigen and antibody.
2. is always the same as avidity.
3. is an expensive imported automobile.
4. is a measure of the strength of antigen-antibody binding.
5. Answers 2 and 4 are correct.
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Pregnancy Testing
1. Bioassay (Rabbit Test)
Assay for human chorionic gonadotrophin
3. ELISA
2. Agglutination inhibition
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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