Professional Documents
Culture Documents
RobertJ.Boackle,Ph.D.
AntigenAntibodyReactions
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
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
Antigenic determinant 1
Antigenic determinant 2
PROTEIN
ANTIGEN
Antigenic determinant 3
Antigenic determinant 4
Y
Y
Y
Immune Complexes
Excess Antibody
Y
Y Y Y
Y
Y
Y
Y Y
Y
Y
ANTIBODY
EXCESS
NO CROSSLINKS
NO Precipitate
Y
Y
Y
Y
Y
Y
Amount of Precipitate
ZONE of
Equivalence
No Soluble Ag or Ab
ANTIGEN
EXCESS
Ab CONC
ANTIBODY
EXCESS
Y
Y
DOUBLE DIFFUSION
Antigen
Antibody
Immune Complex
Antigen
Antibody
Antigen
Immune Complexes
Zone of Equivalence
Rabbit Serum
as antigens
1:4
1:20
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
(Molecule #1)
Antigen 2
(Molecule #2)
OUCHTERLONY ANALYSIS
Partial - Identity
Antigen 3
Antigen 4
is a part
of antigen 4
This animal
was only
injected with
Antigen #4
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
Antigen 5
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
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
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
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
Y
Y
IgM >>IgG