Immunology Lecture
Robert J. Boackle, Ph.D.
Antigen-Antibody Reactions
Specific Objectives: THE STUDENT SHOULD BE ABLE TO
1. Discuss immunoglobulin variability (ie. the variable region)
2. Describe bonds between the variable region and the antigenic
determinant
3. Define antibody affinity and antibody avidity
4. 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)
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.
ANTIGENIC DETERMINANTS
INTERACT WITH
SPECIFIC ANTIBODY
IgG has a Valence of 2
TWO Identical ANTIGEN BINDING SITES
CH2
CH3
CH2
CH3
Surface
of an
Antigen
i.e.
bacterial
cell surface
Movement
at the
Hinge
Region
IgG
CH2
CH3
CH2
CH3
Non-Covalent Interactions
Ball in glove fit
VL
Antigenic
Determinant
VH
Charge-Charge Interactions
Hydrophobic Interactions - And good fit !
VL
+
VH
Gene rearrangements and
Mutational Hot Spots
+
-
VL
-+
VH
+
-
Antibody Affinity
Y
Antigenic determinant 1
Antigenic determinant 2
PROTEIN
ANTIGEN
Antigenic determinant 3
Antigenic determinant 4
MUST HAVE POLYCLONAL ANTIBODY
and at least two different antigenic determinants
TO CROSS-LINK PROTEIN ANTIGENS
Y
Y
Y
Immune Complexes
Excess Antibody
Y
ANTIBODY
EXCESS
Y
Y Y Y
Y
Y
Y
Y Y
Y
Y
NO CROSSLINKS
NO Precipitate
Excess Antigen = Not enough
Cross-links to cause a Precipitation
Y
More cross-links, and higher individual affinities
= higher AVIDITY of the Immune Complexes
Y
Y
Y
ZONE of
Equivalence
No Soluble Ag or Ab
ANTIGEN
EXCESS
Ab CONC
ANTIBODY
EXCESS
Repeating Antigenic Determinants
e.g. PEPTIDOGYCAN
CHO ANTIGENS may cross-link with MONOCLONAL Ab
Y
Y
Y
Y
DOUBLE DIFFUSION
Antigen
Antibody
Immune Complex
Antigen
Antibody
Antigen
Immune Complexes
Zone of Equivalence
Rabbit Serum
as antigens
1:4
1:20
Goat anti-rabbit serum
(Antibodies to rabbit serum)
Non-Identity
Antigen #1
Antigen #2
No Shared Antigenic
Determinants
Antigen #1
Antigen #2
OUCHTERLONY ANALYSIS
Diffusion of Antigens and Polyclonal Antibodies
Non-Identity
Antigen 1
Antigen 2
(Molecule #1)
(Molecule #2)
Antibodies to both antigens
The same Animal was injected with
antigen 1 and with antigen 2
OUCHTERLONY ANALYSIS
Partial - Identity
Antigen 3
This animal
was only
injected with
Antigen #4
Antigen 4
is a part
of antigen 4
Also remember that this
antibody is a multiclonal antibody such as
an anti-serum to an
antigenic preparation
Antibody
Remember that
Protein Antigens
have different
antigenic
determinants
OUCHTERLONY ANALYSIS
Partial - Identity
Antigen 3
Antigen 4
Antibody
Partial - Identity
Antibodies to determinants c and d are only on Antigen 3 and
they pass by antigen 4
Antigen 3
Antibodies
polyclonal antibody
Antigen 4
OUCHTERLONY ANALYSIS
Identity
These two Antigens are the Same Molecule
Antigen 6
is Antigen 5
Antigen 5
No spikes were formed
because:
Antibody
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
Fluorescence
UV Light
Antigens on Cells or on Tissue Sections
Fluorescence
Double layer
Sandwich
UV Light
Antigens
ELISA
Enzyme Linked Immuno-Sorbant Assay
Peroxidase Enzyme is permanently attached to the
Antibody Probe
Substrate that turns
from clear to green
Ag
Ag
Microtiter ELISA
Antigens are immobilized to the plastic surface of a
Microtiter Plate
ELISA
Enzyme Linked Immuno-Sorbant Assay
Peroxidase Enzyme is permanently attached to the
Antibody Probe
Substrate that turns
from clear to green
Ag
Ag
Microtiter ELISA
Antigens are immobilized to the plastic surface of a
Microtiter Plate
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
Agglutination
IgM >>IgG