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Immunology Lecture Robert J. Boackle, Ph.D.
Antigen-Antibody ReactionsSpecific Objectives: THE STUDENT SHOULD BE ABLE TO1. Discuss immunoglobulin variability (ie. the variable region)2. Describe bonds between the variable region and the antigenic determinant3. Define antibody affinity and antibody avidity4. Describe a precipitin curve and discuss lattice formation involving proteins verses carbohydrate antigens and be able to define "zone of equivalence".5. Understand immunodiffusion in agar gels. (identity, nonidentity and partial identity) 6. Have a conceptual understanding of immunoelectrophoresis, Fluorescent antibody techniques and ELISA (enzyme-linked immunoassay) 7. Define "agglutination" and understand the functional differences between monomeric Ab (ie. IgG) and polymeric Ab (ie. IgM and S-IgA)
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Definitions:1. The "antibody affinity" of an antibody-antigen reaction is related to the strength of attractiveness between an antibody (Fab region) and its antigenic determinant.2. The "antibody avidity" is the total strength of binding of the Fab regions of the population of antibodies evoked to an antigen, and involves the reaction with all the antigenic determinates. Thus it is the total strength of the binding of antibodies to antigens. 3. Immune Complex = Antigen-Antibody Complex [the size depends on the ratio of antigen to antibody].
Also the student should be prepared to answer and discuss the following:1. List and describe the possible bonds between the immunoglobulin variable region and an antigenic determinant. Then draw and explain a precipitin curve and "lattice formation" involving protein antigens and polyclonal Ab.2. What is meant by "hypervariable regions" on immunoglobulins? How do B cell clones differ in regard to the hypervariable regions of the immunoglobulins on their surface? At the level of the gene, explain what is believed to account for these clonal diversities.3. Can two different classes of immunoglobulins have identical variable regions? In your answer include a discussion of the switch mechanism.
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ANTIGENIC DETERMINANTS
INTERACT WITH
SPECIFIC ANTIBODY
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CH2 CH3
CH2 CH3
IgG has a Valence of 2
TWO Identical ANTIGEN BINDING SITES
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Movement at the Hinge
Region
CH2 CH3
CH2 CH3
IgGSurface
of an Antigen
i.e. bacterial
cell surface
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Non-Covalent Interactions
Ball in glove fit
Antigenic Determinant
VL
VH
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-
Gene rearrangements and
Mutational Hot Spots
Charge-Charge Interactions
Hydrophobic Interactions - And good fit !
+-
VL
VH
++
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Y
Antibody Affinity
-
+-
VL
VH
++
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Antigenic determinant 1
Antigenic determinant 2
Antigenic determinant 3
Antigenic determinant 4
PROTEINANTIGEN
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Y
YY
MUST HAVE POLYCLONAL ANTIBODYand at least two different antigenic determinants
TO CROSS-LINK PROTEIN ANTIGENS
Immune Complexes
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YYY
YANTIBODYEXCESS
NO CROSSLINKSNO Precipitate
YYY
Y
Y YY
Y
Excess Antibody
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Y
Excess Antigen = Not enough Cross-links to cause a Precipitation
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Y
YY
More cross-links, and higher individual affinities
= higher AVIDITY of the Immune Complexes
YY
Y
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Am
ou
nt
of
Pre
cip
itat
e
Ab CONC
ANTIBODYEXCESS
ANTIGENEXCESS
ZONE ofEquivalence
No Soluble Ag or Ab
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Repeating Antigenic Determinants
e.g. PEPTIDOGYCAN
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CHO ANTIGENS may cross-link with MONOCLONAL Ab
YY Y
Y
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Antigen Antibody
DOUBLE DIFFUSION
Immune Complex
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AntigenAntibody Antigen
Immune Complexes
Zone of Equivalence
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Rabbit Serum
as antigens
1:4 1:20
Goat anti-rabbit serum
(Antibodies to rabbit serum)
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Non-Identity
Antigen #1 Antigen #2
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No Shared Antigenic Determinants
Antigen #1 Antigen #2
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OUCHTERLONY ANALYSISDiffusion of Antigens and Polyclonal Antibodies
Antigen 1(Molecule #1)
Antibodies to both antigensThe same Animal was injected with
antigen 1 and with antigen 2
Antigen 2(Molecule #2)
Non-Identity
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OUCHTERLONY ANALYSIS
Antigen 3is a part of antigen 4
Antibody
Antigen 4
Partial - Identity
Remember that Protein Antigens have different antigenic determinants
Also remember that this antibody is a multi-clonal antibody such as an anti-serum to an antigenic preparation
This animal was only
injected with Antigen #4
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OUCHTERLONY ANALYSIS
Antigen 3
Antibody
Antigen 4
Partial - Identity
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Antigen 3
Antibodiespolyclonal antibody
Antigen 4
Partial - IdentityAntibodies to determinants c and d are only on Antigen 3 and they pass by antigen 4
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OUCHTERLONY ANALYSIS
Antigen 5
Antibody
Antigen 6 is Antigen 5
Identity These two Antigens are the Same Molecule
No spikes were formed because:
Antigenic determinants on Antigen 5 captured all the
antibodies to Antigen 6 and antigenic determinants on Antigen 6 captured all the
antibodies to Antigen 5
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Antigens on Cells or on Tissue Sections
UV Light
Fluorescence
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FluorescenceFluorescenceDouble layer Sandwich
UV Light
Antigens
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Ag
Peroxidase Enzyme is permanently attached to the Antibody Probe
Microtiter ELISAAntigens are immobilized to the plastic surface of a
Microtiter Plate
Enzyme Linked Immuno-Sorbant Assay
ELISA
Ag
Substrate that turns from clear to green
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Ag
Peroxidase Enzyme is permanently attached to the Antibody Probe
Microtiter ELISAAntigens are immobilized to the plastic surface of a
Microtiter Plate
Enzyme Linked Immuno-Sorbant Assay
ELISA
Ag
Substrate that turns from clear to green
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Capture ELISA -- using pre-immobilized mouse monoclonal Ab to capture the Specific Antigen and a second Probe monoclonal Antibody against a different antigenic determinant
Ag Ag
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Agglutination
Y Y
Y
IgM >>IgG